We have cloned, sequenced, and characterized the RNA expression properties of a fish CDKN2 gene from Xiphophorus helleri and X. maculatus. This gene, termed CDKN2X, shows a high degree of amino acid sequence similarity to members of the mammalian CDKN2 gene family, which includes the tumor suppressor loci CDKN2A (P16) and CDKN2B (P15). Comparative sequence analysis suggests that fish CDKN2X is similarly related to all four mammalian gene family members, and may represent a descendant of an ancestral prototypic CDKN2 gene. CDKN2X was mapped to a region on autosomal Xiphophorus linkage group V (LG V) known to contain the DIFF gene that acts as a tumor suppressor of melanoma formation in X. helleri/X. maculatus backcross hybrids. Thus, CDKN2X may be a candidate for the tumor suppressor DIFF gene. Here we have sequenced CDKN2X in both Xiphophorus species and have characterized its expression in normal and melanotic tissues within control and backcross hybrid fish. A simultaneous expressional analysis of the Xmrk-2 tyrosine kinase receptor gene, which is strongly implicated in melanomagenesis in this system, was also performed. RT - PCR analyses revealed that both genes were highly expressed in melanomas. For CDKN2X, this result contrasts numerous findings in human tumors including human melanoma in which either CDKN2A (P16) deactivation or LOH was observed.