Objective To develop a molecular assay for identification of common Culicoides species in Fujian Province based on nested PCR assay, and to investigate the genetic evolution of Culicoides. Methods Culicoides biting midges were captured using light traps in Fujian Province from June 2023 to July 2024 and mounted on microscope slides for morphological identification. The specific primers for nested PCR assay targeting the mitochondrial cytochrome C oxidase subunit 1 (cox1) sequence of Culicoides were designed and synthesized. Genomic DNA was extracted from Culicoides for nested PCR assay and conventional PCR assay, and the amplification products were checked with agarose gel electrophoresis and then compared with morphological dentification results. Following nested PCR assay, the amplification products were sequenced and the resulting reads were assembled using the SeqMan Pro software. The nucleotide homology searches were conducted using the BLAST tool, and the cox1 sequences were aligned using the Clustal X software. The haplotype network was constructed and the haplotype diversity (Hd) index was calculated using the WinArl35 and popart software. The sample sequences were characterized using the MEGA 11 software, and a phylogenetic tree was built using the neighbor-joining method. The intra-population and inter-population genetic distances among different Culicoides species were calculated with P-distance model. Results A total of 35 biting midge samples were collected and morphologically identified as 12 species, including C. arakawai, C. homotomus, C. oxystoma, C. huffi, C. morisitai, C. sumatrae, C. maculatus, C. clavipalpis, C. circumbasalis, C. orientails, Leptoconops chinensis and Lasiohelea wuyiensis. Nested PCR assay successfully amplified specific bands for 29 DNA samples from morphologically identified Culicoides biting midges, with a 100% detection rate of Culicoides (29/29), and conventional PCR assay amplified 17 specific bands with low brightness for 29 DNA samples from morphologically identified Culicoides biting midges, with a 58.6% detection rate of Culicoides (17/29). The BLAST results demonstrated that 4 cox1 sequences of morphologically identified C. circumbasalis had the highest homology (91.64%-91.78%) with the sequence of C. flumineus (ON002367) in Genbank, and had 84.78%-84.87% homology with the reference sequence of C. circumbasalis (KY441782), while 3 cox1 sequences of morphologically identified C. orientails showed the highest homology (91.64%-91.78%) with the sequence of C. flumineus (ON002367) and had 81.93%-82.42% homology with the reference sequence of C. orientails (KT352219). The BLSAT results of other 22 cox1 sequences were consistent with morphological identification results, with sequence homology ranging from 86.78%-100%. The haplotype network diagram of the cox1 gene revealed that the 29 cox1 sequences contained 22 haplotypes, with considerable genetic variation among different Culicoides species (Hd = 0.968 0). Phylogenetic analysis revealed that the 29 cox1 sequences were clustered into the respective clades according to their corresponding subgenera. The inter- and intra-population genetic distances were 0.000 4-0.222 9 and 0.000 0-0.006 2 among different Culicoides species. Conclusion The nested PCR assay-based molecular identification was built, and it could be effectively applied to multiple common Culicoides species in Fujian Province.