[There are three or four themes to Steinbeck's novella; information on these themes can be accessed by clicking on the link to theme that is below this response.]
Arguably, the main theme is that of Alienation. This is a condition that initially results from the historical setting of the Great Depression as men were forced to migrate across the country to wherever they could find work. These itinerant workers were, then, alone in their search for employment, disenfranchised, and alienated from other strangers. That alienation is a major theme in Steinbeck's work is illustrated from the beginning to the end of the narrative. The opening line, for example, connotes this loneliness with the name of the town near which the two isolated men arrive, "A few miles south of Soledad," (Spanish for solitude, grief, isolation).
After George Milton and Lennie Small arrive at the ranch in Chapter Two where they have a job waiting for them, there is caution and mistrust that George exercises about his bunk and towards the old man. George asks him about the previous occupant, and when told that the man was clean, George mistrustfully replies, "I ain't so sure...What did you say he quit for?"(19) Also, in this exchange the tone of alienation is indicated by Steinbeck as "the old man" and "old swamper" are used to describe Candy, rather than his name; it is not until George begins to trust Candy that the man's name is used. And, as another example of George and Lennie's aloneness, the boss, as he walks to the door after questioning the men, "turned and looked for a long moment at the two men." After he leaves, the old swamper comes in and George is suspicious of his having listened when the boss talked to them.
"I didn't hear nothing you guys was sayin'. I ain't interested in nothing you was sayin'. A guy on a ranch don't never listen nor he don't ast no questions." (24)
The old swamper, Candy, is also a lonely and alienated man; he worries that he will be fired because he has lost a hand and can do nothing more that sweep and clean the bunkhouse. Then, later in the chapter Curley, the son of the boss, comes in search of his wife. He, too, is alienated from the others because he is the son of the boss; his wife is the only woman around and she dresses provocatively, causing the men anxiety because the jealous Curley may think that they have made overtures toward her or are looking at her. After Curley's hostile looks at Lennie when he enters the bunkhouse for the first time, George is worried about the man's hostility that marginalizes him from the authorities of the ranch, he tells Lennie,
"....I'm scared I'm gonna tangle with that bastard myself. I hate his guts...."(37)
In Chapter Three, the mule skinner, Slim questions George as he and the other men are suspicious of his and Lennie's traveling together as most itinerant men travel by themselves, another thing that sets them apart from the others.
Curley's wife, too, is alienated. Described only as a genitive of her husband, she has no true identity other than as a temptress, an Eve, luring the men with her red nails and lips. She married Curley because he has some wealth, but after discovering that she is so far from anyone with whom she can socialize or anywhere she can see a movie or have entertainment, she feels completely alone. When she comes to the barn after hearing Lennie talking with the stable buck, Candy tells her "Maybe you better go along to your own house now. The lonely woman retorts,
....Think I don't like to talk to somebody ever' once in a while? Think I like to stick in that house alla time?"(77)
Further, in Chapter 5 she pleads with Lennie to talk to her,
"Why can't I talk to you? I never get to talk to nobody. I get awful lonely." (86)
Of course, no one is more marginalized than Crooks, the black "stable buck" because he is made to stay in the barn, segregated from the other "bindle stiffs." So isolated and alienated is Crooks that he acts defensively in a hostile manner toward the white men if they talk to him. He rebuffs Lennie's attempts to be friendly, even taunting him with the possibility of George's not returning from town until he realizes that Lennie is childlike and harmless. Then, he welcomes the opportunity of being with another person. But, when Candy enters, and then Curley's wife, Crooks becomes anxious after she threatens to tell her husband that Crooks has said or done something that he should not,
Mabye you guys better go....I ain't sure I want you here no more. A colored man got to have some rights even if he don't like 'em"(82)
Indicative of the men's loneliness and alienation is the eagerness with which Lennie asks George to recite their plans for a little farm, as well as the eagerness with which Candy requests being able to join into their venture. Even Crooks has some hope of joining the other men in their dream. Unfortunately, however, this promise of fraternity is foiled by Lennie's inadvertent killing of Curley's wife and George's consequent shooting of Lennie to prevent him from going to prison.
Previously, as Candy looks down at Curley's wife, he speaks in anger against her for destroying his chance to have companionship in the future."You done it, di'n'you?...Ever'body knowed you'd mess things up. You wasn't no good." (95) Previously, George expresses his and Candy's desolation,
George said softly, "--I think I knowed from the very first. I think I knowed we'd never do her. He usta like to hear about it so much I got to thinking maybe we would" (94)
The narrative ends with the solitary George, who "shivered and looked at the gun, and then threw it from him...near the pile of old ashes" (106), walking back with Slim to the ranch, now without his one friend.