The central theme of Rifles for Watie is Jefferson Davis Bussey's transformation from an inexperienced sixteen-year-old into a seasoned gentleman and soldier. At the novel's outset, Jeff displays the bravery required to stand up to the bushwhackers who attack his family's farm, the initiative to journey to Fort Leavenworth and enlist in the Union Army, and the confidence to make independent judgments about the people he encounters and the issues of the era. Although Jeff is unfamiliar with military life, his experiences during the Civil War are profoundly educational. As a foot soldier, artilleryman, cavalryman, scout, and inadvertently a spy, he excels in each position. Through these roles, he realizes that people and issues are more complicated than he initially believed; not all Union soldiers earn his admiration, and he is surprised to find himself feeling loyalty toward personal friends, even when they are officially his adversaries. Initially eager to confront the enemy, Jeff soon understands that war is not a noble pursuit. Each interaction with a Confederate soldier or supporter reveals to him that he and his supposed foes share many similarities. By the end of the Civil War, Jeff returns to his family's farm, having learned significant lessons about human nature and himself.

The novel also delves into the essence of noble conduct. Jeff aspires to be a gentleman and is dismayed to discover that Confederate sympathizers view Union soldiers similarly to how he perceives the bushwhackers. Always polite, particularly to the Confederate civilians he encounters, Jeff earns the respect of others through his kindness. When Union soldiers pause to rest and dine at the opulent home of a Confederate family, the Washbournes, Jeff learns that both Mr. Washbourne and his son, Lee, are part of a rebel cavalry unit led by the notorious General Stand Watie, who orchestrates attacks on Union sympathizers' homes. Despite this, Jeff uses his farming knowledge to assist the Washbourne women by milking their cow and later teaching them how to persuade the cow to accept her calf. Although he fights courageously and kills Confederate soldiers without hesitation in battle, Jeff refuses to join the Union firing squad that executes Lee Washbourne. He demonstrates both compassion and noble character by arranging for Lee's body to be returned to his family for burial.

In stark contrast to the hero's noble nature, Captain Asa Clardy, who leads Jeff's unit, is vindictive, cowardly, cruel, greedy, and deceitful. He falsely accuses Jeff of various offenses and labels him a troublemaker. Clardy avoids combat, tries to intimidate Lucy Washbourne, grinds his heel into the eye of a dying Confederate prisoner, and steals rifles from the Union Army to sell to Stand Watie. Furthermore, Clardy is likely responsible for the murder of Sparrow, a soldier who witnessed one of Clardy's previous acts of theft and murder. By exposing Jeff's identity to Watie's men, Clardy intentionally endangers Jeff's life. Jeff's father, Emory, mentions that Clardy once "had the makings of a good officer," but became embittered after failing to be elected colonel of the Mississippi Volunteer Rifles during the Mexican War.

A more relatable antagonist is the young and spirited Lucy Washbourne, who describes herself as "a rebel—to the backbone." One year younger than Jeff, she rivals him in independence, bravery, honor, loyalty, and even innocence. From their initial encounter, when his dog chases her cat, it's clear they are on opposing sides of every issue; yet, they grow to respect and admire each other. After Jeff reluctantly joins Watie's army, Lucy encourages him to switch sides but does not betray him when he confides that he must return to Fort Gibson with information about the stolen rifles.