Abstract The genus Hepatozoon represents one of six genera in the hemogregarine group. Some studies in snakes indicated effects in the host, from slight influences on fitness to severe effects on growth rate, reproduction and offspring survival rates. Diagnosis and identification are usually through blood smear analyses; but not all infected animals show parasitemia. Based on this, the present study established an adapted molecular protocol to identify Hepatozoon spp. to be used as a complementary test for routine diagnoses at the Clinical Analysis Laboratory at São Paulo Zoological Park Foundation. The study was conducted with 113 individuals. Microscopical analysis and molecular techniques were used to identify the parasite. Microscopic analyses showed 13.3% of the samples to be positive. The first pair of primers, targeting 18S rRNA gene, amplified parasite DNA in 6.3% of the samples. The second pair of primers, targeting Apicoplast fragment, were used only on samples that were identified microscopically as being positive, detecting the presence of parasite DNA in 93.3% of these. Phylogenetic analysis of the resulting sequences found five clusters for the 18S gene and five clusters for the Apicoplast fragment. Studies involving Hepatozoon spp. are still scarce and limited, mainly in snakes and the impacts of this parasite on the vertebrate host, so diagnostic studies are essential for wildlife conservation, especially in ex situ work.