The demonstration of the chromosomal mode of sex determination via genetic experiments as well as the absence of heteromorphic sex chromosomes affirm poeciliid fishes as a unique group among vertebrates that are endowed with the most primitive form of sex chromosomes. In many different taxa the evolutionary process involved in the differentiation of advanced sex chromosomes is outlined through sex specifically organized repetitive sequences. In this investigation hybridization of synthetic probes specific to genomic simple repeat motifs uncovers a sex-specific hybridization pattern in certain viviparous fishes of the family Poeciliidae. The hybridization pattern together with specific staining of the constitutive heterochromatin by C-banding reveals heterogamety in males (Poecilia reticulata) as well as in females (P. sphenops). In P. velifera, however, C-banding alone fails to unravel the heterogametic status. The female specific W-chromosome can be detected by simple repetitive sequence probes. Therefore, the principal significance of heterochromatization as a means of generating differentiated sex chromosomes is evident.