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Phlebotomine sand flies are tiny, hairy, blood-sucking nematoceran insects that feed on a wide range of hosts. They are known as a principal vector of parasites, responsible for human and animal leishmaniasis worldwide. In Thailand, human autochthonous leishmaniasis and trypanosomiasis have been reported. However, information on the vectors for <i>Leishmania</i> and <i>Trypanosoma</i> in the country is still limited. Therefore, this study aims to detect <i>Leishmania</i> and <i>Trypanosoma</i> DNA in field-caught sand flies from endemic areas (Songkhla and Phatthalung Provinces) and non-endemic area (Chumphon Province) of leishmaniasis. A total of 439 sand flies (220 females and 219 males) were collected. Head and genitalia dissection of female sandflies were done for morphology identification, and the remaining parts of those sand flies were then used for the detection of <i>Leishmania</i> and <i>Trypanosoma</i> parasites. The DNA was extracted from individual female sand flies. Polymerase chain reaction (PCR) anneal, specific to the <i>ITS1</i> and <i>SSU rRNA</i> gene regions, was used to detect <i>Leishmania</i> and <i>Trypanosoma</i> DNA, respectively. The positive PCR products were cloned and sequenced. The results showed that the female sand fly species in this study consisted of <i>Sergentomyia khawi</i> (35.9%); <i>Se. anodontis</i> (23.6%); <i>Phlebotomus betisi</i> (18.6%); <i>Ph. kiangsuensis</i> (9.5%); <i>Ph. asperulus</i> (6.4%); <i>Se. barraudi</i> (2.3%); 0.9% of each <i>Se. indica</i>, <i>Ph. stantoni</i>, and <i>Ph. major major</i>; and 0.5% of each <i>Se. sylvatica</i> and <i>Ph. mascomai</i>. The PCR and sequence analysis were able to detect <i>Leishmania</i> and <i>Trypanosoma</i> DNA in sand fly samples, which were identified as <i>L. martiniquensis</i>, 1/220 (0.45%) in <i>Se. khawi</i>, 3/220 (1.36%) of <i>T. noyesi</i> in <i>Se. anodontis</i>, and <i>Ph. asperulus.</i> Fourteen (6.36%) of the unidentified trypanosome species in <i>Se. khawi</i>, <i>Se. indica</i>, <i>Se. anodontis</i>, <i>Ph. asperulus</i>, and <i>Ph. betisi</i> were found in all of the areas of this study. Interestingly, we found a 1/220 (0.45%) co-infection sample of <i>L. martiniquensis</i> and <i>Trypanosoma</i> in <i>Se. khawi</i> from Songkhla Province. These data indicate that several species of sand flies might be potential vectors of <i>Leishmania</i> and <i>Trypanosoma</i> parasites in southern Thailand. However, more extensive study for potential vectors using a larger number of sand flies should be conducted to prove whether these sand flies can be natural vectors of leishmaniasis and trypanosomiasis in both humans and animals. In addition, our study could be useful for the future study of infection prevention, including effective vector control for leishmaniasis and trypanosomiasis in Thailand.