| Cobitoidea, with many diversified species, is one of the most important groups within Cypriniformes. In the present study, we analyzed the evolution of three mitochondrial genes, COI, ND4, and ND5, and four nuclear genes, RH1, RAG1, EGR2B, and IRBP in Cobitoidea species. Based on sequence variations of these genes, we subsequently reconstructed phylogenetic relationships of the major clades for Cobitoidea. The main results are as following.1. The 35 sequences of seven genes of 32 Cobitoidea species were obtained through PCR amplification, cloning and sequencing techniques, with additional 92 sequences of 29 other Cobitoidea species downloaded from GenBank. The distances and variation analyses of these genes were conducted with MEGA 4.0 software. A wide array of models implemented in PAML were used to infer the evolution of these genes leading to some interesting results. (1) For variable sites and parsimony information sites, the mutation of mitochondrial genes was generally larger than that of nuclear genes among all analyzed genes, while. ND5 contains the most variable sites, and EGR2B has the least ones. (2) For base composition, the AT contents (51.3%～57.5%) were higher than that of GC (42.5%～48.7%) in COI, ND4, ND5 and IRBP genes; while the AT contents were almost equal to that of GC in RH1 gene, and the AT (47.6%,39.5%) contents were lower than that of GC (52.4%,60.4%) in RAG1 and EGR2B genes. (3) Selective pressure analysis and site inspections suggested that each gene utilized in the present study were affected by negative selection. COI gene was influenced by the strongest selection pressure, while IRBP gene the weakest. Bayesian identification showed one site of the RH1 gene was significantly under positive selection which located in the trans-membranes domain of protein region encoded by RH1 gene. (4) Branch-model likelihood ratio test showed that the differences of interspecific evolutionary rateωin mitochondrial COI, ND4 and ND5 genes in Cobitoidea were significant, while that in nuclear genes not. This means that each mitochondrial gene presented obvious interspecific heterogeneity due to the progress of independent molecular evolution, while the nuclear genes, which played more significant roles in inheritance and development, were more conservative. 2. Based on Bayesian analysis (BA) and Maximum Likelihood method, we analyzed the phylogenetic relationship of Cobitoidea using the mitochondrial gene COI, ND4+5 and nuclear gene RH1, RAG1, EGR2B and IRBP as molecular markers. Three operational datasets were employed:(1) using a single gene only, (2) using combined mitochondrial DNA sequences and combined nuclear DNA sequences, respectively, (3) using DNA sequences of combined both mtDNA and ncDNA data, which leading to 6 datasets 12 phylogenetic trees,2 datasets 4 trees, and one dataset in 2 trees respectively. Relationships of taxa derived from Bayesian analyses and ML method based on combined analysis to all sequences showed that five families of Cobitoidea, Balitoridae, Nemacheilidae, Cobitidae, Botiidae, and Vaillantellidae were monophyly with robust supports. The construction of molecular phylogenetic relationship based on combined datasets of both mtDNA and ncDNA sequences performed better than that based on single genes only. The present study support that Balitoridae and Nemecheilidae were sister group; Vaillantellidae as a separate lineage. In conclusion, we proposed that the phylogentic relationship of Cobitoidea as: (Botiidae+(Vaillantellidae+(Cobitidae+(Nemacheilidae+Balitoridae)))).
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