We evaluated performance characteristics of five diagnostic methods for cutaneous leishmaniasis. Patients who came to the Leishmania Clinic of Hospital Nacional Cayetano Heredia in Lima, Peru, were enrolled in the study. Lesion smears, culture, microculture, polymerase chain reaction (PCR), and leishmanin skin test (LST) were performed. A total of 145 patients with 202 lesions were enrolled: 114 patients with 161 lesions fulfilled criteria for cutaneous leishmaniasis. Sensitivity and specificity were 57.8% (95% confidence interval [CI] = 50.2-65.4%) and 100.0% for culture, 78.3% (95% CI = 71.9-84.7%) and 100.0% for microculture, 71.4% (95% CI = 64.4-78.4%) and 100.0% for smears, 78.2% (95% CI = 70.6-85.8%) and 77.4% (95% CI = 62.7-92.1%) for LST, and 96.9% (95% CI = 94.2-99.6%) and 65.9% (95% CI = 51.4-80.4%) for PCR. PCR was more sensitive than the other assays (P < 0.001). Sensitivities of culture, smears, and LST varied by lesion duration and appearance. PCR offers performance advantages over other assays, irrespective of patient age, sex, lesion duration, or appearance. That clinical factors influence performance of non-molecular assays offers clinicians a patient-focused approach to diagnostic test selection. Copyright © 2010 by The American Society of Tropical Medicine and Hygiene.