We sequenced nine introns of 25 silkworm (Bombyx mori L.) strains, assuming that the introns are particularly prone to mutation. Mean sequence divergence and maximum sequence divergence in each intronic sequence among 25 silkworm strains ranged from 0.81% (3.8 nucleotides) ~ 9.15% (85.2 nucleotides) and 1.2% (seven nucleotides) ~ 39.3% (366 nucleotides), respectively. The degree of sequence divergence in some introns is very variable, suggesting the potential of using intronic sequences for strain identification. In particular, some introns were highly promising and convenient strain markers due to the presence of a large indels (e.g., 403 bp and 329 bp) in only a limited number of strains. Phylogenetic analysis using the individual or the nine concatenated intronic sequences showed no clustering on the basis of known strain characteristics. This may further indicate the potential of the intronic sequences for the identification of silkworm strains.