The London Story (Murder of John Openshaw):
* John Openshaw: A man who flees America to England, fearing for his life. He is murdered by a mysterious assailant.
* Sherlock Holmes: The brilliant detective who investigates Openshaw's murder.
* Dr. Watson: Holmes' faithful companion and narrator of the story.
* Inspector MacDonald: Scotland Yard detective who initially investigates Openshaw's murder.
The American Story (The Valley of Fear):
* McMurdo: A seemingly charming stranger who arrives in the American mining town of Vermissa, later revealed to be the mastermind behind the "Valley of Fear."
* Ettie Shafter: A young woman who is drawn to McMurdo's charm but later becomes a target of his machinations.
* "Boss" McGinty: The ruthless leader of a secret criminal organization known as the "Scowrers" in Vermissa.
* "Black" William: A member of the Scowrers and one of the key figures in the organization.
* "The Old Man" (Edward Lyon): A retired detective who survived the "Valley of Fear" and helps Holmes solve the mystery.
* Father Ignatius Gallaher: A Catholic priest in Vermissa who is aware of the Scowrers' criminal activities and assists Holmes.
* Cecil Barker: A young man who is caught in the crossfire of the Scowrers' activities and becomes a key figure in the investigation.
Supporting Characters:
* Mrs. Hudson: The landlady at 221B Baker Street, Holmes' and Watson's residence.
* Mr. Douglas: A newspaper editor who provides information about the Scowrers.
* The Colonel: A military officer who becomes involved in the investigation.
The novel seamlessly blends the two storylines, revealing how the events in the Valley of Fear are connected to Openshaw's murder in London.