The series initially starred veteran movie supporting actor Ward Bond as the wagon master, later replaced upon his death by John McIntire, and Robert Horton as the scout, subsequently replaced by lookalike Robert Fuller a year after Horton had decided to leave the series. The series was inspired by the 1950 film Wagon Master directed by John Ford and starring Ben Johnson, Harry Carey Jr. and Ward Bond, and harkens back to the early widescreen wagon train epic The Big Trail starring John Wayne and featuring Bond in his first major screen appearance playing a supporting role. Horton's buckskin outfit as the scout in the first season of the television series resembles Wayne's, who also played the wagon train's scout in the earlier film.

Ballard's trail jumpers attack the Wyatt Company wagon train, killing young John's parents and kidnaping his brother, Jim. In post-Civil War California, John Wyatt, now a man, pulls together a vigilante posse, The Singing Riders, who all ride white horses, dress alike, and ride the trails singing and rounding up outlaw gangs. Meanwhile, John is ever on the lookout for the gang that murdered his parents As a youngster John Wyatt saw his parents killed and his brother kidnapped. On a wagon train heading West he meets his brother who is now a spy for the gang which originally did the dirty work. He and his brother both fall for Mary Gordon When Ballard and his men attack the Wyatt wagon train, they kill all except two young brothers. Twelve years later one brother John has organized a vigilante group. The other brother Jim is now part of Ballard's gang and the two are destined to meet again

The pioneering trail to Oregon was littered with constant danger. Yet, the hope of the "promised land" keeps American families westward bound despite overwhelming odds. A calm, clear-thinking pioneer attempts to lead a wagon train through territory occupied by Pawnees and Sioux. Along the way, the hardy settlers face horse thieves, kidnappers, and unpredictable Indian attacks in their push to establish a new life in the rugged West.