Cutaneous leishmaniasis cases have increased dramatically in recent years in Nepal. The study offers molecular identification of the Leishmaniaspecies using 40 patient's aspiration biopsy samples, targeting markers kinetoplast minicircle DNA (kDNA) and internal transcribed spacer-1 (ITS1). Among molecularly diagnosed 22 cutaneous leishmaniasis cases, L. donovani complex was identified in 13 instances and L. major in 9 cases. The ITS1 PCR was positive in 12 of the positive nested- kDNA PCR cases (12/22), confirming L. donovani complex in seven of the cases and L. major in five of the cases. In addition, the study conclude that concurrent occurrence of atypical cutaneous infections caused by L. donovani parasite in 59.1% of cases and typical cutaneous infections caused by L. major parasite in 40.9% of cases. The detected L. donovani species present null genetic distances from seven references of L. donovani, but slight differences between ITS1 sequences and not grouped into a significant monophyletic cluster indicating the possibility of new variant of or hybrid circulating in the country.