Archaeologists say the skulls were unearthed in August.
Holes drilled into the skulls indicate they were once strung together on a skull rack called tzompantli, commonly used during ancient religious rituals.
According to University of Florida archaeologist Susan Gillespie, the findings reveal novel information on the way the pre-Colombian civilization used and reused skulls for ritual events.
The grouping of skulls is the largest number ever found in one offering.
Experts stumbled upon the skulls while renovating a section of the sacred temple that dates back more than 500 years.
Mexico's National Institute of Anthropology archaeologist Raul Barrerra said they believe the 45 skulls were those of women and men between the ages of 20 and 35.
The Mexican government also announced the finding of a sacrificial stone, a sacred tree, the skeleton of a young woman, and 200 jaw bones last August.
According to Barrerra, the sacrificial rock was the key in the discovery.
"Underneath the sacrificial stone, we found an offering of five skulls. These skulls were pierced with a stick," he said. "These are very important findings."
The different findings which laid hidden for 500 years also challenged the common belief that a person being sacrificed was killed by cutting open the chest and pulling out the heart.
"We normally associate (it) with heart removal rather than decapitation," said Gillespie. "It ultimately gives us a better understanding of how the Aztecs used the human body in various ways in their ritual practices.