Canine hepatozoonosis, caused by Hepatozoon canis, is a tick-borne disease in domestic and wild dogs that is transmitted by ingestion of Rhipicephalus sanguineus ticks. The aim of the study was to detect H. canis in stray dogs in Iran with the use of blood smear examination and molecular techniques. From October 2014 to September 2015, 150 EDTA blood samples were collected from stray dogs in northeastern Iran. Blood smears were microscopically examined for the presence of Hepatozoon gamonts; whole blood was evaluated by PCR with subsequent sequencing and phylogenetic analysis. Hepatozoon spp. gamonts were observed in the neutrophils of 5/150 (3.3%) blood smears, whereas Hepatozoon spp. 18S rDNA was detected in 12/150 (8.0%) blood samples from stray dogs. There was a good correlation between microscopy and PCR methods (κ = 0.756). The highest seasonal rate of infection was detected in the summer (P < 0.05). The frequency of Hepatozoon spp. infection did not differ significantly by gender or age (P > 0.05). The alignment analysis of the sequenced samples showed ≥99% similarity with other nucleotide sequences of Hepatozoon spp. in GenBank. The phylogenetic tree also revealed that the nucleotide sequences in this study were clustered in the H. canis clade and different from the Hepatozoon felis and Hepatozoon americanum clades. Accordingly, we conclude that H. canis infection is present among dogs in northeastern Iran.