BACKGROUND: Biting midges of the genus Culicoides play an important role in the transmission of pathogenic arboviruses and parasites. Thailand has documented more than 100 species of Culicoides; however, several cryptic species complexes remain to be clarified. Recent studies in areas with leishmaniasis indicate that several species of Culicoides might be potential vectors of Leishmania in the subgenus Mundinia, but evidence supporting the hypothesis is still lacking. Therefore, the diversity of Culicoides biting midges and their potential role as vectors of leishmaniasis in southern Thailand remains uncertain. METHODS: Female Culicoides biting midges were collected using Centers for Disease Control and Prevention (CDC) ultraviolet (UV) light traps from four locations within leishmaniasis-affected areas in three provinces of southern Thailand, including Nakhon Si Thammarat, Krabi, and Surat Thani. Culicoides species were identified based on the morphology of wing spot patterns and subsequently confirmed by cytochrome c oxidase subunit I (COI) Sanger sequencing. A potential cryptic species was classified using an integrative taxonomic approach associated with DNA barcoding identification by Barcode of Life Database (BOLD) and Basic Local Alignment Search Tool (BLAST) searches. Furthermore, three different methods of species delimitation, namely ASAP [Assemble Species by Automatic Partitioning], TCS [Templeton, Crandall, and Sing], and PTP [Poisson Tree Processes], were employed to verify the sequences into the molecular operational taxonomic unit (MOTU). Detection of Leishmania and other trypanosomatid parasites was performed by polymerase chain reaction (PCR) based on the ITS1 region and small subunit SSU ribosomal RNA (rRNA) gene, followed by Sanger sequencing and haplotype diversity analysis. The identification of host blood sources was carried out using host-specific multiplex PCR. RESULTS: A total of 716 unfed midges and 159 blood-fed specimens were morphologically identified into 25 species belonging to five subgenera (Avaritia, Hoffmania, Meijerehelea, Remmia, and Trithecoides) and four species groups (Clavipalpis, Ornatus, Shermani, and Shortti). Two unidentified specimens were classified into two subgenera (Trithecoides and Avaritia). The DNA barcoding identification exhibited an 82.20% success rate. Species delimitation analyses demonstrated the presence of cryptic species complexes, categorized into six species: Culicoides actoni, C. orientalis, C. huffi, C. palpifer, C. clavipalpis, and C. jacobsoni. Furthermore, 6.42% of the Culicoides biting midges tested positive for Leishmania DNA in three sampling sites in Nakhon Si Thammarat and Surat Thani provinces (with no positive results in Krabi province). Furthermore, the sympatric infection of Leishmania martiniquensis and Leishmania orientalis was identified in several Culicoides species in Ron Phibun and Phunphin districts in Nakhon Si Thammarat and Surat Thani, respectively. In contrast, L. orientalis was detected in Sichon district, Nakhon Si Thammarat province. A genetic diversity analysis revealed high haplotype diversity and relatively low nucleotide diversity in both parasite populations. Additionally, Crithidia sp. and Crithidia brevicula were detected in Culicoides peregrinus and Culicoides subgenus Trithecoides. The analysis of the host blood meal from Ron Phibun also demonstrated that Culicoides had fed on cows, dogs, and chickens, and mixed blood preferences for humans and cows or chickens and cows were detected. CONCLUSIONS: The findings of the present study demonstrate the presence of mixed blood hosts and co-circulation of L. martiniquensis and L. orientalis in Culicoides in areas of leishmaniasis, as well as cryptic species of Culicoides biting midges, through an integrative taxonomic approach. These findings support the hypothesis that Culicoides biting midges may serve as potential vectors in southern Thailand, and vector diversity is a contributing factor to the risk of zoonotic transmission.