Linkage between an autosomal regulatory gene Diff controlling differentiation of neoplastic melanophores and an esterase locus, Est-1, has been demonstrated in Xiphophorus. The recombination values between Est-1 and Diff ranged from 21 to 29 percent depending on the melanophore pattern loci Sp, Sd, Sd, and Livar. The average recombination frequency for all genotypes investigated was 25 percent. Since Est-1 and Diff do not assort independently of each other, it is concluded that Est-1 and Diff are both located on the same chromosome. Thus, the Est-1 locus provides a marker for monitoring the presence of the Diff autosome.