The Stampede's Chuckwagon Safety Commission says the latest horse was hurt in a collision between two rigs on Monday night.
The horse was euthanized on Tuesday after a veterinary consultation with its owner, B.J. Carey.
A horse was also euthanized on Saturday night after breaking a leg during the chuckwagon races.
On Tuesday, a national animal-rights organization called Animal Justice called on the Calgary Humane Society to prosecute "inhumane rodeo practices" at the Calgary Stampede.
The group says chuckwagon racing is so dangerous that more than 50 horses have been killed during the event at the Stampede since 1986.
“The democratically enacted laws of Alberta unequivocally state that it is illegal to cause or permit animals to be unreasonably in distress,” says Anna Pippus, director of farmed animal advocacy with Animal Justice.
“The appropriate course of action for a law enforcement body that has grave animal welfare concerns with particular activities is to prosecute those activities for unlawful animal cruelty, not to declare its opposition in policy statements.”
The chuckwagon commission says it has determined both Carey and the driver of the other rig, Shane Nolin, were responsible for the incident.
Since the rules normally stipulate the driver responsible must pay $10,000 to the owner of the animal, the commission says Carey will instead receive only $5,000 from Nolin.
In its news release, the commission calls the deaths "extremely regrettable" and says the Stampede is working to ensure the focus of the drivers "is running a safe, clean race."