Dogs can act as a reservoir of canine leishmaniasis disease, which is caused by Leishmania species. The study aimed to identify and document the genotype of cutaneous leishmaniasis (CL) in the stray dogs in Riyadh Province using kinetoplast DNA (kDNA) as a target gene by using nested polymerase chain reaction (nPCR). This cross-sectional investigation was conducted over the course of two years, from March 2016 to July 2018, in different districts of Riyadh Province, Saudi Arabia. A total of 237 dogs were examined, only 18 of the dogs were suspected clinically of cutaneous leishmaniasis due to the presence of cutaneous nodules and cutaneous lesion. Biopsy tissue collections were performed and DNA was extracted. CSB2XF and CSB1XR primers were used to amplify the Leishmania kDNA regions. The Leishmania species were detected by specific 13Z and LIR primers by applying nested PCR assay. Nine dogs were found to be positive for Leishmania major. The examined dogs were negative for other Leishmania spp. The phylogenetic analysis and blast results of kDNA showed that the 9 isolates L. major is closely related (99.9%) to the L. major isolate CMG_irfan5, accession number HQ727556.1 from human, Pakistan. This is the first molecular study on dog leishmaniasis from Saudi Arabia confirmed that dogs have a L. major infection. Further epidemiological and molecular investigations are required to study domestic and wild canine infections with L. major and other Leishmania spp in endemic and nonendemic areas of Saudi Arabia as part of leishmaniasis control