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The tick-borne pathogens of the genus Hepatozoon affect domestic animals and wildlife; their prevalence has risen around the world in the past years. In Mexico there is not enough data available about their surveillance. This study aimed to detect the prevalence of Hepatozoon by PCR in domestic animals and ticks from a fragmented rainforest area from southeast Mexico and analyze the phylogeographic structure of the parasites detected. The total prevalence of H. canis in mammals was 9.7% (20/206; 95% Confidence limits: 6.0-14.6%), being dogs the species with the highest prevalence, of 63.3% (19/30; 95% Confidence limits: 43.9-80.1%). The phylogenetic analysis revealed that sequences from this study were closer to the sequence of H. canis of domestic origin, rather than from wild origin, but in an independent cluster. Haplotypes from our study were geographically restricted to Mexico and the closest haplotype was from Brazil. Ticks that resulted positive by PCR were identified as Amblyomma cajennense (A. mixtus) and Rhipicephalus turanicus. Under fragmented and disturbed conditions of habitat in Balancan, the presence of H. canis may represent a potential risk for other species of domestic and wildlife animals. To the knowledge of the authors, this study represents the first molecular finding of H. canis in Mexico in both domestic animals and ticks. This research lays the groundwork for further studies in order to elucidate the relationships between domestic hosts, wildlife and ticks and describe the life cycle of this parasite in the area., (Copyright © 2016 Elsevier B.V. All rights reserved.)