Information on Leishmania species diversity in western Brazilian Amazon and the clinical picture of human cutaneous leishmaniasis it causes is scarce. We describe clinical findings, diagnostic procedures and identification of Leishmania species in patients from that region. methods The sample consisted of 50 patients, prospectively evaluated for epidemiological and clinical characteristics by means of a structured questionnaire. Conventional and molecular tools were applied to confirm the parasitological diagnosis and identify the species responsible for the disease. results Patients were predominantly male (76.5%) and living in rural areas. Median average age was 1 18 years and median average disease evolution was 8 weeks. For the diagnostic procedures of leishmanin skin test, direct visualization of amastigotes in dermal scrapings and parasite culture of aspirates of the ulcer border were positive for 98%, 52% and 34%, respectively. Molecular methods applied to DNA extracted from skin biopsies of the 50 patients yielded 100%, 82% and 44% positivity by PCR minicircle kDNA, PCR-RFLP ITS1rDNA and PCR-glucose-6-phosphate (G6P), respectively. Fourteen samples from 13 patients were successfully isolated and identified. Multilocus enzyme electrophoresis, PCR-RFLP ITS1rDNA and PCR-G6P permitted identification of the Leishmania species responsible for the aetiology of American tegumentary leishmaniasis in 60% of the examined patients: 16 Leishmania (Viannia) braziliensis, 12 Leishmania (Viannia) lainsoni, 1 Leishmania (Viannia) guyanensis and 1 putative hybrid of Leishmania (Viannia) naiffi and L. (V.) lainsoni.