We report the first establishment of in vitro cultivation and genotypic characterization of Leishmania siamensis isolated from an autochthonous disseminated dermal and visceral leishmaniasis in a Thai acquired immunodeficiency syndrome (AIDS) patient. The molecular identification has shown that the parasite was identical to L. siamensis, a recently described Leishmania species reported in the southern provinces of Thailand. The phylogenetic analysis has confirmed L. siamensis as closely related to the zoonotic Leishmania species L. enrietti.