ABSTRACT. The complete mitochondrial genome of the Southern catfish (Silurus meridionalis) and the Chinese catfish (S. asotus), was determined using the long and accurate polymerase chain reaction (LA-PCR) method. The mitochondrial DNA nucleotide sequences of S. meridionalis and S. asotus were compared with those of 47 other catfish species in the same order. The total length of mitochondrial DNA for S. meridionalis and S. asotus was 16,526 and 16,525 bp, respectively, and included 13 proteincoding genes, 2 ribosomal RNA genes, 22 transfer RNA genes, and a noncoding control region. This mitochondrial gene arrangement is identical to that observed in other Siluriformes. To determine the relative phylogenetic positions of S. meridionalis and S. asotus, and to discover phylogenetic relationships among 24 families of Siluriformes, analyses were conducted, based on mitochondrial DNA, 12S ribosomal RNA, 16S ribosomal RNA, and 13 protein-coding gene sequence data sets. Phylogenetic analyses were congruent with a basal split of the order into Clupeiformes, Characiformes, Cypriniformes, and Siluriformes, and supported a closer relationship of the Southern catfish (family Siluridae) and the Chinese catfish (family Siluridae) to Pimelodidae than to Bagridae. We concluded that these two species are part of a molecular clade that is different from that proposed in recent studies, in which Amblycipitidae appears as a sister group. Our results showed Amblycipitidae appearing as the most basal extant, and Bagridae appearing as a sister group of Cranoglanididae and Pangasiidae. The Siluriformes showed close phylogenetic relationship to the Characiformes.