Background: Cutaneous leishmaniasis (CL) is generally diagnosed by molecular methods, including PCR, using biopsy samples, skin scrapings and clinical exudates. In this study, we assessed the PCR performance for diagnosis of CL using skin of biopsy samples vs PCR of skin lesion exudate samples on filter paper and compared the diagnostic concordance of PCR using both sampling methods. Methods: We assessed the PCR performance using 80 skin biopsy samples and 80 filter paper samples containing exudates from skin lesions obtained from 74 patients with clinical suspicion of CL in Cusco, Peru. Results : PCR using skin biopsy samples had superior diagnostic accuracy compared with filter paper PCR (62.5% [50/80] vs 38.7% [31/80], respectively; p<0.005) and the diagnostic concordance between both samplingmethods was 'moderate' (kappa coefficient=0.50, 95% CI 0.98 to 1.0). Conclusion: PCR using biopsy samples remains the standard for diagnosis of CL.