The wall of a major blood vessel leading away from the heart weakened and then burst, causing blood to fill the horse's abdomen, Stampede officials said in a release Friday afternoon.
"The left lead horse experienced a ruptured aortic aneurysm, leading to sudden internal bleeding and explains why the horse faltered, weakened and then collapsed," said chief veterinarian Greg Evans.
The rupture took place in the abdomen, "which explains the hind limb weakness noted in the horse prior to collapse."
Evans said the pre-existing condition is undetectable in animals and could have ruptured at any time during exercise.
When the horse collapsed, it caused a crash that injured two others so badly they had to be euthanized on the track. A fourth horse needed surgery and is expected to survive.
Driver Chad Harden was thrown into the air along with an outrider. Neither of the men was hurt.
Harden, who tearfully told reporters late Thursday he was devastated by the crash, has been told of the autopsy results.
Toxicology and drug testing will also be done to find out if there were any other factors that could have led to the horse's death.
The autopsy results will be used to determine possible improvements to the Stampede's Fitness to Compete program, in which veterinarians inspect the horses before they compete.
"The Calgary Stampede, and the people who bring their animals to the Stampede, care deeply about the welfare and well-being of those animals. The Stampede works collaboratively to mitigate risks to human and animal competitors," the release said.
An animal welfare group said earlier Friday it wants the chuckwagon races at the Stampede stopped.
"We think that all the changes that the Calgary Stampede has been publicizing over the past few months that were supposed to make this race safe obviously haven't worked," said Peter Fricker, a spokesman for the Vancouver Humane Society.
"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."
Fraser said the Vancouver Humane Society is an "activist group with an activist agenda" that has always pushed for the end of chuckwagon races.
"We rely on the advice from heavy animal experts. We don't rely on the advice from organizations like VHS," said Fraser.
"Although people have the right to divergent opinions, VHS is certainly not the expert on animal care with respect to large animals."
The Calgary Humane Society has taken a different tack than its Vancouver counterpart when it comes to animal welfare at the Stampede.
It's against using animals for entertainment, but acknowledges the chuckwagon races and rodeo events aren't going away.
"We think that working with the Stampede is the best that we can do to help make these events as safe as possible," said spokeswoman Christy Thompson.
She says humane society officers are on the grounds for all events and are there to help the Stampede make "educated changes" to enhance safety.
"I believe honestly that the Calgary Stampede has worked diligently to make these events as safe as possible."
The Stampede introduced some new rules last year aimed at making chuckwagon races safer for both horses and competitors. The move came after six horses died in 2010.
All horses are now inspected by veterinarians when the animals arrive at the Stampede and before and after every race. There is also a mandatory rest day after every four days of racing.
The number of outriders that accompany each chuckwagon as it thunders around a dirt track was reduced to two from four to try to avoid congestion. Several riders have been seriously injured over the years.