Due to the presence of genetically well-defined sex chromosomes, with a relatively restricted sex-determination region containing markers identified at the molecular level, the platyfish Xiphophorus maculatus is one of the best models for the positional cloning of a master sex-determining gene in fish. Both male and female heterogametes and three different types of sex chromosomes have been described in the platyfish, with several loci involved in pigmentation, melanoma formation, and sexual maturity closely linked to the master sex-determining locus. Using the melanoma-inducing oncogene Xmrk, its protooncogenic counterpart egfrb, as well as other X- and Y-linked molecular markers, bacterial artificial chromosome (BAC) contigs have been assembled for the sex-determining region of X. maculatus, which was mapped by fluorescent in situ hybridization to the subtelomeric region of the sex chromosomes. Initial sequence analysis of these contigs revealed several gene candidates and uncovered syntenies with different mammalian and chicken autosomes, supporting an independent origin of sex chromosomes in platyfish and tetrapods. Strikingly, the sex determination region of the platyfish is very instable and frequently undergoes duplications, deletions, and transpositions. This instability might be linked to the high genetic variability affecting sex determination and other sex-linked traits in Xiphophorus.