We analyzed 3633 Japanese Bombyx mandarina COI sequences from 37 locations in Japan, found 94 segregating sites in 714-bp long COI nucleotide sequences and eventually identified 121 haplotypes. We further confirmed that the 121 haplotypes were different from the 8 B. mori haplotypes reported by Yukuhiro et al. (2011), and the result strongly suggested little gene flow from B. mori to Japanese B. mandarina. We discuss the properties of the mutational differences between the two species.