The single intron of the heavy-chain fibroin gene in domesticated (Bombyx mori) and wild (B. mandarina) silk moths has a length of approximately 1000 nucleotides. It is located only 57 bp from the gene's core promoter and harbors multiple AT-rich regulatory elements that have been found to enhance the basal level of transcription in vitro. In this work, the intronic nucleotide variability among members of both Bombyx species is analyzed. The intron sequences of B. mori strains k120 and Nistari, as well as B. mandarina specimens from Japan and Korea, were obtained. This information was compared with the previously reported sequences of B. mori strains p50 and C-108, as well as an additional B. mandarina specimen collected in Japan. We found a total of 26 variant positions, including variants shared by members of both species and species-specific changes. The potential functional role of these variants was investigated by using the program MatInspector to search for putative binding sites of transcription factors within the intron. We detected a multitude of putative binding elements distributed along the entire intronic sequence. Among them, 22 correspond to protein binding domains that are known to regulate fibroin transcription. The mapping of multiple variant positions within these putative binding sequences as well as in known regulatory elements of the intron argue for functional significance on the regulation of transcription.