The genus Hepatozoon consists of apicomplexan protozoans that affect mammals, birds, amphibians, and reptiles. In dogs, the Hepatozoon species include H. canis and H. americanum, which are transmitted by the Rhipicephalus sanguineus tick and cause nonspecific signs, such as fever, weight loss, diarrhea, and blood disorders. These protozoans have a worldwide distribution in Europe, Asia, Africa, and North and South America, including Colombia. The aim of this study was to determine the prevalence and risk factors associated with H. canis in the urban and rural areas of Ibagué, Colombia. Blood samples were collected from 308 dogs (180 rural areas and 128 urban areas). Collected data included dog breed, sex, age, environmental factors, and the presence of ectoparasites. A fragment of the 18S rRNA gene was amplified by PCR for detection of the pathogen and confirmed by sequencing. Among the 308 samples, 14 were positive (14/308, 4.5%) for the presence of H. canis. The partial sequence of the 18S rRNA gene showed identity values >98% with H. canis, forming a cluster with sequences from Latin America. An epidemiological survey found two protective factors: most of the time at home (P=0.055) and overnight stay at home (P=0.03). This is the first molecular study of the prevalence and phylogeny analysis of H. canis in Ibagué, Colombia. The findings may help determine risk factors and enhance our understanding of the geographic distribution of H. canis in Colombia.