Bombyx mori and Bombyx mandarina are morphologically and physiologically similar. In this study, we compared the nucleotide variations in the complete mitochondrial (mt) genomes between the domesticated silkmoth, B. mori, and its wild ancestors, Chinese B. mandarina (Ch Bm) and Japanese B. mandarina (Ja Bm). The sequence divergence and transition mutation ratio between B. mori and Ch Bm are significantly smaller than those observed between B. mori and Ja Bm. The preference of transition by DNA strands between B. mori and Ch Bm is consistent with that between B. mori and Ja Bm, however, the regional variation in nucleotide substitution rate shows a different feature. These results suggest that the Ch Bm mt genome is not undergoing the same evolutionary process as Ja Bm, providing evidence for selection on mtDNA. Moreover, investigation of the nucleotide sequence divergence in the A+T-rich region of Bombyx mt genomes also provides evidence for the assumption that the A+T-rich region might not be the fastest evolving region of the mtDNA of insects.