Skin biopsies stored in ethanol from 49 patients with suspected cutaneous leishmaniasis (CL) were tested in a real-time polymerase chain reaction (PCR) assay and compared with conventional diagnostic methods. With clinical diagnosis as the gold standard, PCR had a sensitivity of 96% (47/49) vs. 61% (30/49) for histopathology and 33% (16/49) for culture. In addition, DNA was extracted from 70 frozen smears of lesions from suspected cases of CL and tested with the same assay. In these samples, the PCR had a sensitivity of 61% (43/70) vs. 56% (39/70) for histopathology and 41% (29/70) for culture. In this study, real-time PCR offered a rapid diagnosis with an enhanced sensitivity over conventional methods. Although the yield of PCR diagnosis was lower when testing frozen smears, the assay still outperformed existing diagnostic modalities.