Leishmania siamensis is newly described as the causative pathogen of autochthonous leishmaniasis in Thailand. Potential vectors and animal reservoirs of L. siamensis are not thoroughly studied. An environmental survey was carried out in the affected area in two provinces in southern Thailand: Songkhla and Nakhon Si Thammarat. Ninety-nine villagers, 378 sandflies, and potential animal reservoirs were examined. Leishmania DNA amplicon was identified in two species of female sandflies, Sergentomyia (Neophlebotomus) and Sergentomyia (Parrotomyia) barraudi. The DNA amplicon was also identified in black rats (Rattus rattus). A phylogenetic tree of confirmed patients, sandflies and black rats fell into a single clade and separate from other Leishmania species. This study showed the potential involvement of R. rattus and Sergentomyia (Neophlebotomus and Parrotomyia) sandflies in transmission of L. siamensis.