Mending Fences was euthanized on the track after breaking down in the Dixie Stakes, a preliminary to the Preakness, which was Barbaro's downfall a year ago.
A 5-year-old stallion with seven wins in his career, Mending Fences sustained a compound fracture of his right front leg.
"The horse was put down," Pimlico vice president Mike Gathagan said.
A green screen was put up in front of the stallion to shield the procedure from a stunned crowd of more than 100,000.
Mending Fences was leading as he approached the end of the backstretch, with less than a half mile to go in the turf race. Suddenly, he tumbled over headfirst, throwing jockey Eddie Castro.
Another horse, Einstein, threw its jockey, Robby Alborado, and continued around the track without a rider while medical personnel raced to the scene of the fallen horse and the two riders. Both jockeys were able to walk away; neither appeared to be seriously hurt, though they rode off in an ambulance.
Alborado was set to ride Curlin, one of the top contenders in the Preakness.
The breakdown was another blow to a sport still reeling from the death of Barbaro, who was euthanized in January after extensive treatment failed to save him from a devastating injury seconds after the start of last year's Preakness.
The fatal injury to Mending Fences came in the race that followed the inaugural Barbaro Stakes, which was supposed to bring a bit of closure to last year's heartbreak. Barbaro's owners, Roy and Gretchen Jackson, were on hand to present a crystal bowl in the winner's circle.