Like mammals, Lepidoptera possess female-specific sex chromatin. In a compilation of new and published data, 81% of the 238 investigated Lepidoptera species display one or more heterochromatin bodies in female somatic interphase cells, but not in male cells. In contrast with the similar phenomenon in mammals, this sex-specific heterochromatin does not function as a dosage compensation mechanism. Most Lepidoptera have a WZ/ZZ sex chromosome mechanism, and the sex chromatin is derived from the univalent W sex chromosome. Sex chromatin is regarded as an indicator of an advanced stage of W chromosome evolution. In species with a Z/ZZ sex chromosome mechanism, loss of the W chromosome is accompanied by loss of the female-specific heterochromatin. Since sex chromatin can be discerned easily in interphase nuclei, and especially so in the highly polyploid somatic cells, it is a useful marker for diagnosing chromosomal sex of embryos and larvae, and of identifying sex chromosome aberrations in mutagenesis screens. All species with sex chromatin belong to the Ditrysia, the main clade of Lepidoptera that contains more than 98% of all extant species. Sex chromatin has not been reported for clades that branched off earlier. The nonditrysian clades share this character with Trichoptera, a sister group of the Lepidoptera. We propose that Lepidoptera originally had a Z/ZZ sex chromosome mechanism like Trichoptera; the WZ/ZZ sex chromosome mechanism evolved later in the ditrysian branch of Lepidoptera. Secondary losses of the W chromosome account for the sporadically occurring Z/ZZ sex chromosome systems in ditrysian families. The lepidopteran sex chromatin, therefore, appears to mirror the full evolutionary life cycle of a univalent sex chromosome from its birth through heterochromatinization to sporadic loss.