| Summary: | Over the years, cases of autochthonous leishmaniasis have been dramatically increasing in Thailand. Recently, several publications have claimed certain species of the phlebotomine sand flies and biting midges potentially serve as natural vectors of <i>Leishmania</i> and <i>Trypanosoma</i> species in this country. However, more information regarding the vector–parasite relationships, as well as their natural reservoirs in the country, still needs to be explored. Herein, we hypothesized that synanthropic reptiles in the leishmaniasis-affected area might be a natural reservoir for these parasites. In this present study, a total of nineteen flat-tailed house geckos were collected from the house of a leishmaniasis patient in Songkhla province, southern Thailand, and then dissected for their visceral organs for parasite detection. Small subunit ribosomal RNA (<i>SSU rRNA</i>) gene and internal transcribed spacer 1 (<i>ITS-1</i>)-specific amplifications were conducted to verify the presence of <i>Trypanosoma</i> and <i>Leishmania</i> parasites, respectively. Only <i>Trypanosoma</i> DNA was screened positive in eight gecko individuals by <i>SSU rRNA</i>-PCR in at least one visceral organ (4, 4, and 6 of the heart, liver, and spleen, respectively) and phylogenetically related to the anuran <i>Trypanosoma</i> spp. (An04/Frog1 clade) previously detected in three Asian sand fly species (<i>Phlebotomus kazeruni</i>, <i>Sergentomyia indica</i>, and <i>Se. khawi</i>). Hence, our data indicate the first detection of anuran <i>Trypanosoma</i> sp. in the flat-tailed house geckos from southern Thailand. Essentially, it can be inferred that there is no evidence for the flat-tailed house gecko (<i>Hemidactylus platyurus</i>) as a natural reservoir of human pathogenic trypanosomatids in the leishmaniasis-affected area of southern Thailand.
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