There are several themes in Winesburg, Ohio, by Sherwood Anderson. As listed with eNotes, the three themes are "rite of passage," "isolation and alienation" and "doubt and ambiguity."
A "theme" is defined as:
...a...repeated idea that is incorporated throughout a literary work...a thought or idea the author presents to the reader....
The theme that speaks most strongly to me is that of "alienation and isolation." The two characters in which this theme is most clearly seen are Enoch Robinson and Elizabeth Willard—George Willard's mother. Epoch Robinson is alienated not because of what he has experienced in this small town, but by what he experienced in New York before he moved to Winesburg.
Elizabeth Willard's sense of isolation comes from a feeling that others in her past knew her and her predicament in terms of the life she leads in Winesberg and cared about her. These were men who traveled through town and boarded at the Willard home.
They seemed to understand and sympathize with her.
In "Mother," she is now connected to no one. She removes herself to her room, rejecting the company of others. Her alienation is mostly of her own making, though one does not sense that it brings her happiness or satisfaction. This alienation includes Elizabeth's son, George.
In "Death," Elizabeth almost connects with Dr. Reefy, but when they are interrupted as they hug one another, the tenuous bond is broken, even though Dr. Reefy is in love with Elizabeth.
Doctor Reefy did not see the woman he had held in his arms again until after her death.
While the theme of "rite of passage" deals more distinctly with George Willard—the story's main character—the theme that seems most unsettling and impactful to me is that of "alienation and isolation."