Leishmaniasis is a vector-borne parasitic disease caused by protozoa of the genus Leishmania, transmitted primarily through the bites of infected female sand flies (Phlebotomus spp. in the Old World and Lutzomyia spp. in the New World). It presents in several clinical forms, including cutaneous, mucocutaneous, and the visceral form known as kala-azar, the latter being the most severe. Dogs are considered a primary reservoir for Leishmania, the main causative agent of zoonotic visceral leishmaniasis (ZVL), contributing to the human-animal transmission cycle. This study aimed to diagnose the disease by this protozoan molecular and microscopically in stray and domestic dogs using a staining by gimsa stain. In the current study, a total of 75 blood samples of dogs (different in sex and age) were collected from other areas of Babylon City during the period from December 2024 to February 2025. The microscopic findings found that dogs infection by leishmania donovani the infection rate 41.33% (31 out of 75). A molecular study using PCR and sequencing techniques identified gene of leishmania donovani in dog sample. The sequencing technique demonstrated the species of L.donovani in this study. The molecular results demonstrated that 41 out of 75 dogs (54.66%). According to sex, no significant difference (p =0.669) was found in the infection, where it was 53.19% (25 out of 45) in males, while 57.14% (16 out of 28) in females. For the age groups, there was no significant difference (p=0.073), which has a high incidence of illness in canines less than 1 years 22/34(64.70%) compared to other age groups. Current data also revealed that the infection rate in rural areas was slightly higher than in urban areas, where it recorded 15/19 (78.94%) and 12/19 (63.15%), respectively, with significant differences (P = 0.025).