- James Tyrone Sr. – 65 years old. He looks ten years younger and is about five feet eight inches tall but appears taller due to his military-like posture and bearing. He is broad-shouldered and deep-chested and remarkably good-looking for his age with light brown eyes. His speech and movement are those of a classical actor with a studied technique, but he is unpretentious and not temperamental at all with "inclinations still close to his humble beginnings and Irish farmer forebears". His attire is somewhat threadbare and shabby. He wears his clothing to the limit of usefulness. He has been a healthy man his entire life and is free of hang ups and anxieties except for fear of "dying in the poorhouse" and obsession with having money. He has "streaks of sentimental melancholy and rare flashes of intuitive sensibility". He smokes cigars and dislikes being referred to as the "Old Man" by his sons.
- Mary Cavan Tyrone – 54 years old, the wife and mother of the family who lapses between self-delusion and the haze of her morphine addiction. She is medium height with a young graceful figure, a trifle plump with distinctly Irish facial features. She was once extremely pretty and is still striking. She wears no make-up and her hair is thick, white and perfectly coiffed. She has large, dark, almost black, eyes. She has a soft and attractive voice with a "touch of Irish lilt when she is merry". Mary has been addicted to morphine since the difficult birth of her youngest son Edmund. The doctor who treated her simply gave her painkillers, which led to a longtime morphine addiction that continues to plague her.
- James "Jamie" Jr. – 33 years old, the older son. He has thinning hair, an aquiline nose and shows signs of premature disintegration. He has a habitual expression of cynicism. He resembles his father. "On the rare occasions when he smiles without sneering, his personality possesses the remnant of a humorous, romantic, irresponsible Irish charm – the beguiling ne'er-do-well, with a strain of the sentimentally poetic". He is attractive to women and popular with men. He is an actor like his father but has difficulty finding work due to a reputation for being an irresponsible, womanizing alcoholic. He and his father argue a great deal about this. Jamie often refers to his father as "Old Gaspard", a character from the opera Les cloches de Corneville, who is also a miser.
- Edmund – 23 years old, the younger and more intellectually and poetically inclined son. He is thin and wiry. He looks like both his parents but more like his mother. He has her big dark eyes and hypersensitive mouth in a long narrow Irish face with dark brown hair and red highlights from the sun. Like his mother, he is extremely nervous. He is in bad health and his cheeks are sunken. Later he is diagnosed with tuberculosis. He is politically inclined to have socialist leanings. He traveled the world by working in the merchant navy and caught tuberculosis while abroad.
- Cathleen – "The second girl", she is the summer maid. She is a "buxom Irish peasant", in her early twenties with red cheeks, black hair and blue eyes. She is "amiable, ignorant, clumsy with a well-meaning stupidity".
Several characters are referenced in the play but do not appear on stage:
- Eugene Tyrone – A son born before Edmund who died of measles at the age of two. He was infected by Jamie who was seven at the time and had been told not to enter his room but disobeyed. Mary believes that Jamie had the intent of hurting Eugene.
- Bridget – A cook.
- McGuire – A real estate agent who has swindled James Tyrone in the past.
- Shaughnessy – A tenant on a farm owned by the Tyrones.
- Harker – A friend of James Tyrone, "the Standard Oil millionaire", owns a neighboring farm to Shaughnessy with whom he gets into conflicts, often postulated to be based on Edward Harkness, Standard Oil heir, who had a summer home nearby in Waterford, Connecticut.
- Doctor Hardy – The Tyrones' physician at the insistence of James, though the other family members don't think much of him and suspect he is only their doctor because he is cheap.
- Captain Turner – The Tyrones' neighbor.
- Smythe – A garage assistant whom James hired as a chauffeur for Mary. Mary suspects he is intentionally damaging the car to provide work for the garage.
- The mistress – A woman with whom James had had an affair before his marriage, who had later sued him causing Mary to be shunned by her friends as someone with undesirable social connections.
- Mary's father – Died of tuberculosis.
- James's parents and siblings – The family immigrated to the United States when James was 8 years old. His father abandoned the family two years later and returned to Ireland, where he died after ingesting rat poison. It was suspected suicide but James refuses to believe that. He had two older brothers and three sisters.
