Leishmania infantum parasite is the main causative agent of visceral leishmaniasis (VL) which threatens a wide range of humans and dogs in Iran. Our objective was to investigate Leishmania parasite species in Ardebil Province a well-known VL foci targeting ITS-rDNA gene, sequencing and phylogenetic analysis. Vacuum EDTA tubes were used for blood collection of asymptomatic dogs using non-invasive methods in Ardabil, northwestern Iran in August 2019. Phenol-chloroform DNA extraction, ITS-rDNA gene PCR and sequencing were performed. Leishmania species was accurately identified based on sequences and Maximum likelihood phylogenic analyzes. 11 of 31 asymptomatic dogs were definitively infected with L. infantum. Under 5 years old age group had the highest infection rate, which showed that this group is more sensitive to VL in this area and also the infection was mainly observed in male sheepdogs. These findings show that non-invasive sampling and molecular methods are reliable and convenient in the diagnosis of visceral leishmaniasis. Regarding the 35. 5% infection ratio in the area control and monitoring measures of human individuals and canine reservoirs should be taken to consideration.