A San Antonio Fire Department official said they found "stacks of bodies" and no signs of water in the truck, which was found next to railroad tracks in a remote area on the city's southern outskirts.
Sixteen other people found inside the trailer were transported to hospitals for heat stroke and exhaustion, including four minors, but no children were among the dead, the department said.
"The patients that we saw were hot to the touch, they were suffering from heat stroke, exhaustion," San Antonio fire chief Charles Hood told a news conference. "It was a refrigerated tractor-trailer but there was no visible working A/C unit on that rig."
Temperatures in San Antonio, which is about 250km from the Mexican border, swelled to a high of 39.4 degrees Celsius on Monday with high humidity.
The city's police chief William McManus said a person who works in a nearby building heard a cry for help and came out to investigate. The worker found the trailer doors partially opened and looked inside and found a number of dead bodies.
McManus said this was the largest incident of its kind in the city and said three people were in custody following the incident, though their involvement is not yet clear.
A spokesperson for US Immigration and Customs Enforcement (ICE) said that its Homeland Security Investigations division was investigating "an alleged human smuggling event" in coordination with local police.