Leishmania is a parasitic protozoan which is transmitted to humans through the bite of an infected female Phlebotomus and Lutzomyia sand flies. Cutaneous leishmaniasis (CL), caused by Leishmania major and L. tropica, is an endemic disease in many areas of Jordan and considered as a major public health problem. The political instability in the Syrian Arab Republic has resulted in the immigration of large number of refugees into Jordan where most of them resided in camps near the Syrian borders. Therefore, the main objective of the present study was to inspect Leishmania species/genotypes which are responsible for CL infections among Syrian refugees and compare them with the recovered species/genotypes isolated from Jordanian patients. Three molecular-based assays (ITS1-PCR-RFLP, Nested ITS1-5.8S rDNA PCR, and Kinetoplast DNA PCR) followed by sequencing and phylogenetic analysis were undertaken and compared for their efficiency to confirm CL diagnosis and genotype the infecting Leishmania species. Thereafter, the evolutionary relationships among various Leishmania isolates from Syrian and Jordanian CL patients were elucidated. Results from the present study indicated that 20 and 9 out of the inspected 66 patients (39 Jordanian and 27 Syrian) were infected with L. major and L. tropica respectively. ITS1-PCR RFLP typing proved to be more sensitive in the detection of Leishmania species (positive in 44% of the isolates) compared to both ITS1-5.8S rDNA gene and Kinetoplast DNA PCR which were successful in identifying Leishmania species only in 23% and 33% of the isolates respectively. Sequencing and phylogenetic analysis of ITS1 and ITS1-5.8S rDNA genes revealed high levels of heterogeneity among the sequenced isolates. One sample typed as L. tropica from Jordanian patient showed high similarity with L. tropica sample isolated from a Syrian patient in a Lebanon refugee camp; therefore, the need for comprehensive studies to confirm if any new L. tropica strains might be introduced to Jordan by Syrian refugees is urgently indicated. These observations highlighted the need for further studies to clarify the risk status of species and strains which might be introduced from Syria to Jordan.