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Autochthonous leishmaniasis in Thailand has recently been a public health concern due to an increasing number of new clinical cases. Most indigenous cases were diagnosed with Leishmania (Mundinia) martiniquensis, and Leishmania (Mundinia) orientalis. However, some doubts regarding vector misidentification have arisen and need to be elucidated. Accordingly, we aimed to assess the species composition of sand flies and determine the molecular prevalence of trypanosomatids in the transmission area of leishmaniasis in southern Thailand. In the present study, a total of 569 sand flies were caught from the vicinity of a visceral leishmaniasis patient's house in Na Thawi District, Songkhla Province. Of these, 229 parous and gravid females consisted of Sergentomyia khawi, Se. barraudi, Phlebotomus stantoni, Grassomyia indica, and Se. hivernus, accounting for 31.4%, 30.6%, 29.7%, 7.9%, and 0.4%, respectively. However, Se. gemmea, which has previously been proposed as the most abundant species and putative vector of visceral leishmaniasis, was not found in the present study. Based on ITS1-PCR and sequence analysis, two specimens of Gr. indica and Ph. stantoni showed positive amplification of L. martiniquensis and L. donovani complex, respectively, the first one being presumed indigenous and the second one being not. Anuran Trypanosoma was also molecularly detected using SSU rRNA-PCR and ubiquitously found in 16 specimens of four dominant sand fly species except for Se. hivernus. The obtained sequences could be phylogenetically categorized into the two major amphibian clades (An04/Frog1 and An01+An02/Frog2). The existence of the monophyletic subgroup and distinct lineage suggests them as novel Trypanosoma species. The TCS network analysis of these anuran Trypanosoma sequences also revealed high haplotype diversity (Hd = 0.925 ± 0.050), but low nucleotide diversity (π = 0.019 ± 0.009). Furthermore, the living anuran trypanosomes were microscopically demonstrated in a single specimen of Gr. indica, supporting the vectorial capacity. Importantly, our data confirmed the scarcity of Se. gemmea and also uncovered, for the first time, the co-circulation of L. martiniquensis, L. donovani complex, and suspected novel anuran Trypanosoma spp. in phlebotomine sand files, implicating their potential role as vectors of trypanosomatid parasites. Therefore, the novel data from this study would greatly facilitate the comprehension of the complexity of trypanosomatid transmission and the establishment of prevention and control measures for this neglected disease more effectively., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)