The accident happened in the fourth heat of the GMC Rangeland Derby when the left lead horse on Chad Harden's wagon collapsed in the back stretch.
That brought down the three other horses in the rig, and caused one of the two outriders and his horse to collide with the wagon.
Harden and his outrider were not hurt in the collision.
A necropsy done Friday determined that the first horse died of a ruptured aortic aneurysm near the kidney, said the Stampede's Doug Fraser.
The outrider horse and the right lead horse, both badly injured in the crash, were euthanized by veterinarians.
A third horse, on the right wheel, was also injured but was expected to survive.
Peter Fricker, a spokesman for the Vancouver Humane Society, says there should be a full safety review to find out why the accident happened.
Following the death of four chuckwagon horses in 2010, Stampede officials revamped the rules in a bid to make the sport safer.
The number of outriders was cut from four to two to make the track less congested.
Pre-race health checks by veterinarians and rest days for the horses were also made mandatory.
But Fricker said it's clear now those changes haven't made the sport safer.
"We think that there's something more fundamentally wrong with the race, and we're calling for a suspension of the chuckwagon races and a full and fundamental safety review to be conducted,” he said.
Michael Alvarez-Toye of the Calgary Animal Rights Coalition wants an outright ban on rodeos.
"It always shocks and infuriates me … you'd think after 100 years they'd come to realize they cannot prevent deaths."