Species of the genus Hepatozoon are among the most widespread haemogregarine parasites infecting reptiles; however, information regarding their occurrence in native snakes from Mexico remains scarce. This study investigated the molecular occurrence of Hepatozoon spp. in free-ranging Mexican black-tailed rattlesnakes (Crotalus molossus nigrescens) rescued within the Reserva Ecológica del Pedregal de San Ángel (REPSA), an urban protected area in central Mexico. Opportunistically sampled squirrels were also included to explore the occurrence of these parasites in sympatric mammals. Blood samples from 26 rattlesnakes, six Mexican gray squirrels (Sciurus aureogaster), and three rock squirrels (Otospermophilus variegatus) were screened by PCR targeting a partial fragment of the 18S rRNA gene. Hepatozoon spp. DNA was detected in six rattlesnakes (23.1%) and two Mexican gray squirrels (33.3%), whereas all rock squirrels tested negative. Phylogenetic analyses suggested the presence of two distinct Hepatozoon lineages: one showing phylogenetic affinity with Hepatozoon cf. ophiosauri, detected in all positive rattlesnakes and one squirrel, and another closely related to Hepatozoon griseisciuri, identified in a second squirrel. These findings represent the first molecular detection of Hepatozoon in C. molossus nigrescens, the first record of infection in S. aureogaster, and the first report of a H. griseisciuri-like lineage in Mexico. Although parasite identification was based exclusively on partial 18S rRNA gene sequences, this study expands current knowledge of Hepatozoon diversity in Mexican wildlife and provides important baseline information for future studies integrating molecular, morphological, and ecological approaches to better understand parasite diversity and transmission in protected urban ecosystems.