Abstract: Species delimitation and phylogenetic relationships of the genus Pseudogobio fishes in the subfamily Gobio-ninae remain debated and unresolved. This study used molecular species delimitation methods and morphological analyses to reconstruct phylogenetic relationships of Pseudogobio fishes based on nuclear loci and the classification of this genus in China. The results of Bayesian tree showed that Pseudogobio guilinensis and P. banggiangensis were monophyletic specie, and that P. vaillanti and P. escocinus were polyphyletic species that the former included five Clades A-E and the latter was comprised of two Clades A and B. The analyses of POFAD and Structurama showed that each clade of P. vaillanti and P. escocinus were independent genetic populations, and BP & P analysis strongly supported them as different species. The results of *Beast species tree revealed that P. esocinus Clade B occupied a basal position of Pseudogobio fishes; Clades A and B of P. vaillanti were the sister taxon relationship, and P. vaillanti Clade C was the sister taxon of P. esocinus Clade A, and all of them together were the sister group of P. guilinensis; Clades D and E of P. vaillanti were the sister taxon relationship, which were the sister group of P. banggiangensis. A revised classification of Pseudogobio fishes in China by molecular and morphological evidence as follows: P. vaillanti sensu stricto was limited to P. vaillanti Clades A and B; A resurrection of P. longirostris Mori, 1934 was assigned to P. vaillanti Clade C; Clades D and E of P. vaillanti were cryptic species, and P. guilinensis and P. banggiangensis were valid species, respectively.