Hepatozoon is a genus of hemogregarines constituting the most widespread and common reptile hemoparasite. Although various molecular assessments of these parasites have been conducted in lizards from Africa and Europe, similar studies are needed for South American lizards. Through amplification and sequencing of fragments of the 18S rRNA gene, we assess the prevalence of Hepatozoon parasites in 230 geckos from South America, including the endemic species Hemidactylus agrius, Hemidactylus brasilianus, Lygodactylus klugei, Phyllopezus pollicaris, Phyllopezus periosus, and an exotic species, Hemidactylus mabouia. We found an overall low prevalence of Hepatozoon infection (7/230, 3%) with only 3 of the 6 host species infected with Hepatozoon (Hemidactylus mabouia, P. pollicaris, and P. periosus). Within the 7 infected host samples, 5 genetically distinct lineages of Hepatozoon parasites were identified, only 1 of which was similar to previously published haplotypes. Thus, although prevalence is low, genetically based diversity of Hepatozoon in geckos from South America is very high. Three of these lineages appear basal to 1 of the major clades of Hepatozoon, suggesting that this clade might have originated in South America, and thereby indicating a potential phylogeographic pattern that had not been previously identified. Future studies should assess the distribution and competence of invertebrate hosts in the regions analyzed, and Hepatozoon diversity in other less well-known regions of the world.