ABSTRACT. The endosymbiotic Leishmania RNA virus 1 (LRV1) has been associated with severity and clinical manifestations of American tegumentary leishmaniasis caused by species of the Leishmania ( Viannia ) subgenus. Between and within Leishmania species, and among endemic countries, the prevalence of LRV is highly variable. The LRV virus has not been detected in L. ( V. ) panamensis , the second-most prevalent species in Central America and Colombia. However, no systematic screening of LRV has been conducted in L. ( V .) panamensis , and thus it is still controversial whether this virus is truly absent from the species. We sought to determine the prevalence of LRV1 in L. ( V. ) panamensis clinical strains isolated from patients with cutaneous leishmaniasis (CL), from different geographic areas of Colombia. We analyzed 219 clinical strains; 78% were L. ( V. ) panamensis , 18% were L. ( V. ) braziliensis , and 4% were L. ( V. ) guyanensis . Screening for LRV1 was performed by quantitative reverse transcription–polymerase chain reaction. The LRV1 was detected in 18% (7 of 40) of L. ( V ) braziliensis strains, and was not detected in any of the L. ( V. ) guyanensis or L. ( V. ) panamensis strains. The LRV1-positive L. ( V ). braziliensis strains came from the Amazon Basin. Of the seven LRV1-positive strains, two were isolated from patients with mucocutaneous leishmaniasis, and the remaining from patients with CL. Our results confirm the absence of LRV1 in L. ( V. ) panamensis in Colombia.