In this study, we tested the utility of the mitochondrial gene cytochrome c oxidase subunit 1 (CO1) as the barcode region to deal with taxonomical problems of Polypedilum (Tripodura) non-biting midges (Diptera: Chironomidae). The 114 DNA barcodes representing 27 morphospecies are divided into 33 well separated clusters based on both Neighbor Joining and Maximum Likelihood methods. DNA barcodes revealed an 82% success rate in matching with morphospecies. The selected DNA barcode data support 37–64 operational taxonomic units (OTUs) based on the methods of Automatic Barcode Gap Discovery (ABGD) and Poisson Tree Process (PTP). Furthermore, a priori species based on consistent phenotypic variations were attested by molecular analysis, and a taxonomical misidentification of barcode sequences from GenBank was found. We could not observe a distinct barcode gap but an overlap ranged from 9–12%. Our results supported DNA barcoding as an ideal method to detect cryptic species, delimit sibling species, and associate different life stages in non-biting midges.

P. (T.) anningense sp. n. and P. (T.) biloborum sp. n. are described and illustrated as male imagines from Oriental China. DNA barcodes of two new species are also presented. A key to known male imagines of the subgenus Tripodura from China is given.

The subgenus Tripodura Townes of Polypedilum Kieffer from China including 26 species is reviewed. Eleven new species, named P. (T.) absensilobum Zhang & Wang sp. n., P. (T.) apiculusetosum Zhang & Wang sp. n., P. (T.) arcuatum Zhang & Wang sp. n., P. (T.) bilamella Zhang & Wang sp. n., P. (T.) conghuaense Zhang & Wang sp. n., P. (T.) dengae Zhang & Wang sp. n., P. (T.) mengmanense Zhang & Wang sp. n., P. (T.) napahaiense Zhang & Wang sp. n., P. (T.) parallelum Zhang & Wang sp. n., P. (T.) pollicium Zhang & Wang sp. n. and P. (T.) trapezium Zhang & Wang sp. n. are described and illustrated based on male imagines. Three species, P. (T.) quadriguttatum Kieffer, P. (T.) unifascia (Tokunaga) and P. (T.) udominutum Niitsuma are firstly recorded in China. A key to known male imagines of Chinese species and an updated world checklist of subgenus Tripodura are presented.

Polypedilum (Tripodura) falcatum sp. n. and P. (T.) procerum sp. n. of the acifer species group of Polypedilum subgenus Tripodura Townes are described and illustrated as male imagines from China. The male adult of P. (T.) falcatum sp. n. is distinguished by the presence of two faint markings on wing; short and interrupted abdominal tergite bands; the sickle-like superior volsella bearing 0–2 outer setae and a tuft of short setae on its apex. The male adult of P. (T.) procerum sp. n. differs in having short abdominal tergite bands; a long, slender and apically curved superior volsella; a high fore leg ratio (2.30–2.33). A key to known male imagines of Polypedilum subgenus Tripodura from China is presented.

The genus Harnischia Kieffer, 1921 is recorded for the first time from Mongolia and Harnischia disseta sp. nov. is described and illustrated as male imagines based on specimens collected from Khyargas Nuur. A key to known male species of the genus is presented.

Tanytarsus dawai Lin, sp. nov., within the Tanytarsus norvegicus species group, is described and illustrated as larvae and adults from Xizang, China. The holotype of T. sinuatus Goetghebuer, 1936 is examined and the larvae of T. sinuatus from China (Qinghai, Xizang and Yunnan) are described and illustrated for the first time.