Genetic hybrids of Xiphophorus fishes have been used for decades to study heritable melanoma formation. In these models, overexpression of pigmentation patterns from melanin-producing pigment cells can lead to genetically regulated melanoma formation in backcross hybrids. In the best studied of these models, the Gordon-Kosswig hybrid melanoma, tumors form spontaneously in all individuals of a subset of backcross hybrids between the platyfish Xiphophorus maculatus Jp 163 A and the swordtail species Xiphophorus helleri. Backcross hybrids susceptible to melanoma formation inherit a sex-linked oncogene, Xmrk, associated with the spotted dorsal (Sd) pigment pattern and have lost both copies of an autosomal gene, DIFF, from the X. maculatus parent. Spontaneous melanoma formation conforms to simple, two-gene Mendelian inheritance in which DIFF behaves as a recessive tumor suppressor gene. Recently, Xiphophorus hybrids in which melanomas can be induced by UV and near-UV visible light exposure have been described. We report here results of genetic linkage analysis of one of these Xiphophorus light-inducible hybrid melanoma models, in backcross hybrids between the two platyfish species X. maculatus Jp 163 B and Xiphophorus couchianus. Our linkage results provide the first estimate of recombination between the tumor suppressor locus, DIFF, and glycerate-2-dehydrogenase (GLYDH) in Xiphophorus linkage group V. Also, they demonstrate that DIFF regulates hyperplasia of spotted side (Sp) pigment cells in this hybrid model, analogous to its regulation of hyperplasia of Sd pigment cells in the "classical" Gordon-Kosswig hybrid. Joint segregation analyses of melanoma-bearing fish indicate that segregation of DIFF is genetically linked to melanoma induction by 405 nm light in this model but that induction of melanomas by UV wavelengths apparently does not depend on segregation of the DIFF locus.