We analyzed PCR-amplified carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, and dihydroorotase (CAD) gene fragments from 146 Bombyx mori native strains and found extremely low levels of DNA polymorphism. Two haplotypes were identified, one of which was predominant. CAD haplotype analysis of 42 samples of Japanese B. mandarina revealed four haplotypes. No common haplotype was shared between the two species and at least five base substitutions were detected. This result was suggestive of low levels of gene flow between the two species. The nucleotide diversity (π) scores of the two samples differed markedly: lower π values were estimated for B. mori native strains than Japanese B. mandarina. We further analyzed 12 Chinese B. mandarina derived from seven areas of China, including Taiwan. The results clearly indicated that the π score was ~80-fold greater in Chinese B. mandarina than in B. mori. The extremely low level of DNA polymorphism in B. mori compared to its wild relatives suggested that the CAD gene itself or its tightly linked regions are possible targets for silkworm domestication.