Lepidopteran species have a relatively high number of small holocentric chromosomes (Bombyx mori, 2n = 56). Chromosome identification has long been hampered in this group by the high number and by the absence of suitable markers like centromere position and chromosome bands. In this study, we carried out fluorescence in situ hybridization (FISH) on meiotic chromosome complements using genetically mapped B. mori bacterial artificial chromosomes (BACs) as probes. The combination of two to four either green or red fluorescence-labeled probes per chromosome allowed us to recognize unequivocally each of the 28 bivalents of the B. mori karyotype by its labeling pattern. Each chromosome was assigned one of the already established genetic linkage groups and the correct orientation in the chromosome was defined. This facilitates physical mapping of any other sequence and bears relevance for the ongoing B. mori genome projects. Two-color BAC-FISH karyotyping overcomes the problem of chromosome recognition in organisms where conventional banding techniques are not available.