We newly identified the W chromosome in eight lepidopteran species and confirmed the absence of W chromosome in Caligula japonica females with the Z0 sex chromosome constitution by means of genomic in situ hybridization (GISH). In pachytene oocytes of species with the WZ/ZZ sex chromosome system, female-derived genomic probes of the respective species highlighted the whole W chromosome thread in the WZ bivalent and also stained specific autosomal region(s) with telomeric and/or subtelomeric signals. On the basis of GISH results obtained in this study and earlier published, we classified karyotypes of lepidopteran species into three types.