Due to their phylogenetic position, Psychidae play an important role in the investigation of the W chromosome origin in Lepidoptera. Several species of Psychidae were tested for the presence of sex-chromatin and investigated via comparative genomic hybridization. Furthermore, odd chromosome numbers and a Z univalent were observed in females. Overall, this study brings tangible evidence for the absence of the W chromosome in Psychidae, thus contributes to complex knowledge of the W chromosome evolution. In addition, karyotypes of the given species were analyzed using 18S rDNA and histone H3 probes. The results indicate relative stability of their karyotypes.