Hepatozoon americanum is a protozoan that causes American canine hepatozoonosis (ACH) in the southern United States; Hepatozoon canis, the causative agent of canine hepatozoonosis in Africa, Asia, Europe, and South America, has not previously been definitively identified in dogs in the United States. To characterize the diversity of Hepatozoon spp. in domestic dogs from Oklahoma, blood samples collected from dogs residing in an endemic area of the state, clinical cases presented to veterinarians with symptoms of ACH, and dogs housed at a local shelter were evaluated by a nested PCR designed to amplify a variable region of the 18S rRNA gene of blood ampicomplexa, including Hepatozoon spp. Hepatozoon sequences recovered from a dog from an area where ACH is endemic, from clinically ill dogs, and from one shelter dog most closely resembled H. americanum. However, two other shelter dogs had evidence of infection with H. canis or a closely related organism. A subsequent review of real-time PCR results from the Molecular Diagnostics Laboratory at Auburn University revealed that the majority of samples submitted from dogs from across the United States which tested positive for Hepatozoon spp. had H. americanum. However, some submissions were also found which contained DNA sequence of H. canis. Mixed H. americanum and H. canis-like infections also were detected. Our data suggest that H. americanum, H. canis, as well as H. canis-like organisms are present and may cause disease in dogs in the southern U.S.