Tick-borne diseases have raised serious health concerns in dogs globally, primarily in hot and humid climate of tropics and subtropics. The molecular detection and characterization of Hepatozoon canis were carried out in extracted DNA from blood samples of dogs (n = 212) by real-time PCR and from vector ticks (n = 41) by conventional PCR targeting the 18S rRNA gene. The present study reported low prevalence of Hepatozoon canis infections in dogs (9%) while high prevalence in Rhipicephalus spp. ticks (33.14%) in dogs in eight coastal districts of Odisha. The PCR positive blood samples (n = 19) were analyzed by multiplex PCR for co-infection, revealing the prevalence of co-infections of H. canis with A. platys (2.5%), with both A. platys and E. canis (0.9%) as well as with A. platys and B. gibsoni (0.9%). Presence of ticks, irregular anti- tick treatment and limited outdoor activity were found to be significantly associated incidence of hepatozoonosis. Hematological values of dogs with hepatozoonosis revealed anemia, thrombocytopenia and neutrophilia, while the group with mixed infection (MI) had significantly lower (P < 0.01) mean Hb, platelets, TEC, PCV, MCV, MCH, MCHC level indicating microcytic hypochromic anemia and potential thrombocytopenia in comparison to single infection (SI). The nucleotide sequence identity study revealed 98%-100% similarity of Odisha isolate with other global isolates of H. canis. The Odisha isolate was closely related to the nucleotide sequences of H. canis, isolated from dogs of West indies, as revealed by the phylogenetic analysis.