The mitochondrial DNA cytochrome b gene was sequenced from 27 catfish species representing 11 families and 24 genera catfishes in China. Aligning with cytochrome b sequences of eight catfish species from North America and Africa retrieved from GenBank, and selecting Astyanax mexicanu, Cyprinus carpio, and Sardinops melanostictus as outgroups, we constructed a matrix of 38 DNA sequences. The phylogenetic trees were constructed by using Bayesian method and Maximum Parsimony (MP) method. The results showed that (1) there are three base pair deletions of mitochondrial cytochrome b gene compared with Characiformes, Cypriniformes, and Clupeiformes; (2) the representatives of Chinese catfish species form a monophyletic group; and (3) the molecular phylogenetic trees constructed with both methods suggest that the families Sisoridae, Akysidae and Amblycipitidae form a monophyletic group, and the families Clariidae, Schilbidae, Ariidae, Ictaluridae, Cranoglanididae, Pangasiidae, Siluridae, Claroteidae, and Bagridae also form a monophyletic group. The families Cranoglanididae from China and Ictaluridae from North America form a sister-group relationship, and the families Clariidae, Ictaluridae, Siluridae, and Sisoridae are obviously monophyletic groups. But the position of the family Plotosidae was not resolved by Bayesian analysis and maximum parsimony inference.