We report first-time definitive evidence of human cutaneous leishmaniasis (CL) resulting from L. major infections in Ghana. The evidence is in the form of PCR testing and DNA sequence analysis of skin sample biopsies from residents of the Ho District in the forested Volta Region of southeastern Ghana (Figure 1). The moist semi-deciduous forest of this region of Ghana is atypical for CL. Although two sand fly species with known or suspected potential to transmit L. major were present in communities where human cases of CL originated, they were found in such small numbers that their role in the transmission of CL is uncertain.