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Leishmania RNA virus (LRV) is a double-strand RNA virus that was first detected in members of the Leishmania viannia in the New World. The present study aimed to investigate the presence of LRV in the Leishmania species isolated from cutaneous leishmaniasis (CL) patients and rodents as reservoirs in Isfahan province an old zoonotic CL focus, center of Iran. Totally, 85 samples were collected from CL patients (n = 80) and rodent reservoirs (n = 5) from different regions of Isfahan province. Species identification was determined using the PCR-RFLP method. Viral dsRNA was extracted and for observation of 5.3 kb dsRNA on an agarose gel. The presence of LRV was surveyed using the Semi-nested PCR method. For phylogenetic analyzes, 6 samples of 13 isolates were sequenced and a phylogenetic tree was drawn by MEGA7 version 7.0.26. Of 80 Leishmania isolates recovered from the patients with CL, 79 and only one were identified as L. major and L. tropica , respectively. Also, the PCR assays detected four L. major and one L. turanica in five assessed Rhombomys opimus as the rodent reservoirs. LRV was detected only in Leishmania species isolated from 13 species of 85 (15.3%) CL including (L. major , n = 12) and (L. tropica , n = 1). Phylogenetic analysis showed that they were belonged to LRV2 and had the highest similarity with Iranian reference LRV2 in GenBank. Our results showed that the LRV2 was present in cutaneous Leishmania species in Isfahan province is the most historical and touristic province of Iran. In the study LRV was not reported from rodent reservoirs, it may be due to the small sample size. Phylogenetic analysis of current sequences demonstrated that these isolates belong to the registered LRV2 of the Old World. • Leishmania RNA Virus 2 was detected in 13 (15.3%) of cutaneous leishmaniasis isolates studied. • Leishmania RNA Virus 2 in one L. tropica in Isfahan province, Iran was reported. • Our sequences analysis demonstrated LRV2 isolated from Isfahan are genetically similar to other LRV2 from the Old World [ABSTRACT FROM AUTHOR]