Babesia canis is a severe protozoal illness spread by ticks with worldwide distribution and is associated with anemia, systemic illness, and potentially fatal complications. The present study aimed to investigate the Occurrence of Babesia canis infection in dogs in Babylon Governorate, Iraq, using microscopic and molecular diagnostic methods.100 dog blood samples were collected from of different ages, sexes, health conditions, and management systems. Thin blood smears stained with Giemsa stain were examined microscopically for the detection Babesia spp. In addition, genomic DNA extracted from blood samples and subjected to polymerase chain reaction (PCR) targeting the BC28.1 gene of Babesia canis. The overall prevalence of infection was 48% by microscopic examination and 52% by PCR assay. Microscopically, infected erythrocytes showed characteristic intraerythrocytic pyriform merozoites of B. canis. Clinically infected dogs commonly exhibited fever, pale mucous membranes, anorexia, depression, and signs of anemia. Female dogs showed a higher prevalence of infection (56.8%) than male dogs (42.9%), although the difference was not statistically significant. In contrast, significantly higher prevalence was observed in clinically diseased dogs compared with apparently healthy dogs. The findings of the present study confirm the circulation of Babesia canis among dogs in Babylon Governorate and demonstrate the superior sensitivity of PCR compared with microscopic examination for the diagnosis of canine babesiosis. These findings demonstrate the need for molecular diagnostic tools and sustained epidemiological surveillance programs to enhance the detection, monitoring, and control of the disease.