This study aimed to optimize molecular methods for detecting DNA of Hepatozoonspp. as well as identify the phylogenetic relationships of Hepatozoonstrains naturally infecting domestic dogs in Belém, Pará, northern Brazil. Blood samples were collected from 138 dogs, and screened for Hepatozoonspp. using a new nested PCR assay. Positive samples were subjected to genetic characterization based on amplification and sequencing of approximately 670bp of the Hepatozoonspp. 18S rRNA. Of the positive dogs, four shared the haplotype Belém 01, one dog presented the haplotype Belém 02 and two dogs shared the haplotype Belém 03. A Bayesian inference indicates that haplotypes Belém 01 and Belém 02 are phylogenetically related to H. canis, while Belém 03 is related to H. americanum. Overall, based on the first molecular evidence of H. americanumin Brazilian domestic dogs, the proposed protocol may improve the epidemiological investigation of canine hepatozoonosis.