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Leishmaniasis is a vector-borne disease caused by a protozoan parasite of the genus Leishmania. The infection is transmitted by the bite of infected female sandflies of the genus Phlebotomus in the Old World or Lutzomyia in the New World. The diagnosis is made by stained smear microscopy, in vitro culture, and DNA-based detection methods. The diagnostic methods used in the study included microscopy of Giemsa-stained touch smears, in vitro culture using NNN medium, and ITS1-PCR. The amplicon-based next-generation sequencing based on ITS1 (ITS1-219-NGS) was included in the comparison part of the study. The comparison included two groups: a CL-confirmed group that was positive by any of the tests employed, and a non-CL group that was negative by all diagnostic methods. The results of the ITS1-219-NGS were compared to ITS1-PCR followed by RFLP to identify the parasite species, which is assumed to be the gold standard owing to its superiority over microscopy and in vitro culture. The statistical tools used in the comparison were McNemar’s test, Bhapkar marginal homogeneity test, kappa statistics, and Gart and Buck equations. The comparison showed that ITS1-219-NGS had a higher sensitivity of 94% compared to the assumed gold standard (ITS1-PCR) of 86%. Perfect agreement between the two test methods (kappa statistic = 0.82, 95% CI = 0.73–0.90) was shown. The species agreement between the two tests was fair (Kappa-0.24), with only agreement in 56% of the cases. Furthermore, the standard ITS1-PCR was unable to identify the parasite species in 29 CL cases, but was revealed by ITS1-219-NGS and confirmed by BLAST search. Unlike the ITS1-PCR, the ITS1-219-NGS identified all its positive results correctly, as confirmed by BLAST search. Moreover, 13 ITS1-PCR weak positive CL cases were found to be negative by ITS1-219-NGS, and upon BLAST search shown to be contamination of human and bacterial origin. Amplicon-based ITS1-219-NGS is a superior, feasible, highly sensitive, and high-throughput diagnostic method with accurate species identification that can be used in clinical practice and epidemiologic surveys.