Abstract: In the present study, we cloned and sequenced 10 new ND4 and ND5 gene sequences of Cobitoidea. These sequences were used to reconstruct phylogenetic relationships together with those of 15 other species downloaded from GenBank. The results showed that the length of ND4 gene sequence was 1 380−1 387 bp with ATG as starting codon and incomplete termination signal as terminated codon; the length of ND5 gene sequence was 1 821−1 839 bp with ATG as starting codon and TAA or TAG as terminated codon; three tRNA genes coding tRNAs that carry hisidine, serine and leucine respectively, were inserted between ND4 and ND5 genes. A, T, C and G accounted for 30.4%, 27.3%, 14.2% and 28.1% in ND4/ND5 gene (including intervening three tRNA genes). The content of A+T (57.7%) is higher than that of G+C (42.3%). The estimated Ti/Tv ratio was 1.586. With Danio rerio and Cyprinus carpio as outgroups, the phylogenetic relationships of Cobitoidea were analyzed using maximum parsimony (MP) method, maximum likelihood (ML) method and Bayesian analyses (BI). Results of all the three methods indicated that Cobitinae, Nemacheilinae, Botiinae, Balitoridae and Vaillantellidae were all monophyletic respectively, and their interrelationships were: (Vaillantellidae + (Botiinae + (Cobitinae + (Nemacheilinae + Balitoridae)))), which was consistent with the previous studies based on the whole mitogenome and some nuclear genes sequences (eg. RAG1 gene). However, the present results differ from those based upon other mtDNA genes sequences (e.g. the cyt b and D-loop showed that Nemacheilinae grouped with Cobitinae forming sister-group, then they clustered with Balitoridae). The difference could be due to the phylogenetic information carried by different length sequence: Longer sequences carry more information and result in more reliable phylogenetic trees.