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Background  Phlebotomine sand flies are vectors of  Leishmania  spp. At least 27 species of sand flies have been recorded in Thailand. Although human leishmaniasis cases in Thailand are mainly imported, autochthonous leishmaniasis has been increasingly reported in several regions of the country since 1999. Few studies have detected  Leishmania  infection in wild-caught sand flies, although these studies were carried out only in those areas reporting human leishmaniasis cases. The aim of this study was therefore to identity sand fly species and to investigate  Leishmania  infection across six provinces of Thailand.    Methods  Species of wild-caught sand flies were initially identified based on morphological characters. However, problems identifying cryptic species complexes necessitated molecular identification using DNA barcoding in parallel with identification based on morphological characters. The wild-caught sand flies were pooled and the DNA isolated prior to the detection of  Leishmania  infection by a TaqMan real-time PCR assay.    Results  A total of 4498 sand flies (1158 males and 3340 females) were caught by trapping in six provinces in four regions of Thailand. The sand flies were morphologically classified into eight species belonging to three genera ( Sergentomyia ,  Phlebotomus  and  Idiophlebotomus ).  Sergentomyia iyengari  was found at all collection sites and was the dominant species at most of these, followed in frequency by  Sergentomyia barraudi  and  Phlebotomus stantoni , respectively. DNA barcodes generated from 68 sand flies allowed sorting into 14 distinct species with 25 operational taxonomic units, indicating a higher diversity (by 75%) than that based on morphological identification. Twelve barcoding sequences could not be assigned to any species for which cytochrome  c  oxidase subunit I sequences are available. All tested sand flies were negative for  Leishmania  DNA.    Conclusions  Our results confirm the presence of several sand fly species in different provinces of Thailand, highlighting the importance of using DNA barcoding as a tool to study sand fly species diversity. While all female sand flies tested in this study were negative for  Leishmania , the circulation of  Leishmania  spp. in the investigated areas cannot be ruled out.    Graphical abstractSupplementary Information  The online version contains supplementary material available at 10.1186/s13071-021-04856-6.

Sand fly identification and screening for Leishmania spp. in six provinces of Thailand