Microscopic examination is the standard method for diagnosis of cutaneous and mucocutaneous leishmaniasis despite its low sensitivity. This study compared the diagnosis efficacy of microscopic examination versus polymerase chain reaction (PCR)-based methods and DNA sequencing using whole blood and skin lesion samples from patients with suspected leishmaniasis. The presence of Leishmania was determined by microscopy and amplification of 18S ribosomal RNA gene from blood and skin samples of 22 patients. Twenty individuals were positive for leishmaniasis. Microscopic analysis identified 85%, whereas PCR identified 100% of positive cases from skin and 90% from blood. Cytochrome b gene (cyt-b) amplification and sequencing identified Leishmania guyanensis, Leishmania shawi, and Leishmania naiffi from skin and blood samples. This study demonstrated the usefulness of whole blood and molecular techniques for the diagnosis and species identification of leishmaniasis.