Trypanosoma cruzi, the protozoan causative of Chagas disease, is primarily transmitted through blood-sucking insects and infects mammalian and some reptilian hosts. In Chile, insects of the Mepraia genus are key vectors of T. cruzi in its wild transmission cycle. High prevalence and mixed infection of T. cruzi lineages have been reported in a Mepraia population on Santa María Island in the Atacama Desert. However, no small mammals have been reported. The island’s vertebrate community is dominated by the lizard Microlophus atacamensis and marine and scavenger birds. This study aimed to research blood samples of M. atacamensis for the presence of T. cruzi DNA (kDNA and satDNA) using conventional PCR (cPCR) and quantitative real-time PCR (qPCR) and estimate parasitemia. Our findings reveal that 39.4% of 33 individuals were positive with both cPCR and qPCR, while when assessing infection with either technique, it rises up to 81.8%. These findings confirm that M. atacamensis is a host of T. cruzi, suggesting its potential role as a key reservoir in the island’s transmission cycle. This study provides new insights into the life cycle of T. cruzi in the coastal Atacama Desert, highlighting the importance of reptiles in the epidemiology of this parasite.