There was no collision, but a lead horse for the team of driver Chad Harden suddenly went down, bringing two others horses with him and sending Harden and an outrider flying through the air.
Neither man was hurt but two of the horses had to be euthanized on the track due to their injuries. A fourth horse will require surgery for its injuries but is expected to survive.
Stampede spokesman Doug Fraser said the horse that suddenly collapsed had "experienced a fatal event, the nature of which is undetermined at this time."
In 2010, two horses died of heart attacks during competitions.
Fraser said it went "without saying" that Stampede officials had hoped to complete the 100th anniversary competition without any animal deaths.
"It's a sad situation, but we will investigate the incident to determine what happened in the hopes we can prevent it from happening again," he said.
"These horses are cared for; they are loved by the drivers themselves and there's no question it's a tragedy for these families."
Fraser said it was an emotional night for all Stampede officials.
"Nobody wants to see this happen," he said. "But I think the emotion really showed with Chad Harden. The driver is devastated and even our chief veterinarian ... felt emotional about this.
"We've had absolutely phenomenal success this year, up until tonight."
Accounts of the crash quickly began to surface on Twitter.
"Chad Harding's chuckwagon was turning the third corner of the track and one of his horses went down," wrote John Schuiling.
"He had to make an emergency stop ... one of his outriders and his horse collided into the back of the chuckwagon. They had to temporarily delay the races. I hope all the horses and humans are O.K."
Video of the race showed officials quickly blocking off the crash scene with dark tarp-like material.
The Stampede introduced changes 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.
Last year, the Stampede levied its largest fine ever against driver Cliff Cunningham for a chuckwagon race collision that led to the death of a horse.
The initial fine of $2,500 was upped to $12,500 after a commission reviewed video of the crash and spoke with the drivers involved.
Ironically, Cunningham was the winner of Thursday's night heat.
Despite the Stampede's actions, animal welfare organizations have complained that the rodeo is simply cruelty masquerading as entertainment.
The Vancouver Humane Society, which has noted more than 50 chuckwagon horses have died since 1986, has frequently called for the suspension of the Stampede chuckwagon races.
And this year, prominent animal rights activist and TV game show legend Bob Barker suggested the Stampede celebrate its centenary by shutting down.
"I would like very much to see them celebrate the 100th anniversary of the Calgary Stampede by saying that is enough animal cruelty," Barker said.
"I doubt seriously if they've made any great strides in making rodeo safe. I don't know how they could and I would imagine they talk about it more than they actually do.''