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• Ae. albopictus and "C. pipiens subgroup" were dominant mosquito species in China. • rDNA 28S D5 of Ar. subalbatus and An. sinensis were obtained for the first time. • 28S D5 is a suitable mini-DNA barcode for mosquitoes identification in China. • Two new clades named C. cf. perexiguus and C. new sp were identified in Culex. • Body color, beak, and abdomen were valuable for identifying the Culex clades. Accurate classification and identification of mosquitoes are essential for the prevention and control of mosquito-borne diseases. In this study, adult mosquitoes were collected from 15 cities across 14 provinces in China. They were identified morphologically with the dominant species determined. Furthermore, representative samples were identified at the molecular level based on rDNA 28S D5. In total, 880 adult mosquitoes were collected belonging to Culex (266), Aedes (473), Armigeres (13), and Anopheles (5). Aedes albopictus and " C. pipiens subgroup" were the dominant species. A total of 140 sequences of 28S D5 region (68 for " C. pipiens subgroup", 51 for Ae. albopictus , 18 for Ar. subalbatus , and three for An. sinensis) ranging from 148 to 161 bp were obtained, with 100 % success of amplification and sequencing. Molecular identification were consistent with morphological classification. Sequence analysis showed that " C. pipiens subgroup" was identified into three clades: the traditional C. pipiens subgroup (Clade I), the newly discovered C. cf. perexiguus (Clade II), and C. new sp. (Clade III). Clade I contained the most abundant haplotypes (16) widely distributed without geographical differences. Clade II included six haplotypes that were aggregately distributed south of the Yangtze River. Only three sequences in Clade III showed two haplotypes with no geographical differences. Further morphological comparisons demonstrated differences in body color, beaks, and abdomens among the three clades. In conclusion, the rDNA 28S D5 region could effectively distinguish Culex, Aedes, Armigeres , and Anopheles species at the lower category level, demonstrating its potential as a mini-DNA barcode for mosquito identification. [Display omitted] [ABSTRACT FROM AUTHOR]