The inheritance of melanistic spotting due to the presence of large macromelanophores was investigated in two stocks of eastern mosquitofish of different geographic origin. In one stock, the melanistic phenotype is almost entirely restricted to males end is inherited as a Y-chromosome-linked single-gene trait. The Y-linked melanism allele (M) is temperature sensitive. It is dominant with high penetrance when fish are raised at 22°C. Penetrance is much reduced, to about 42%, when fish are raised under warmer conditions (26°C-29°C). Patterns of sex-linked inheritance observed in this stock imply the existence of an XX-XY sex chromosome system. This is in contrast to the closely related western mosquitofish, which has a WZ-ZZ sex chromosome mechanism. Another stock was founded from a single melanistic female mated to a melanistic male carrying the Y-linked Mallele. Segregation ratios in various crosses are consistent with a model in which melanistic females are a result of atypical sex determination. Melanistic females have an XY sex chromosome makeup but carry a dominant autosomal female-determining gene that overrides the male-determining tendency of the Y chromosome. Thus, melaniatic females, like melanistic males, carry the same Y-linked melanism gene. © 1989 The American Genetic Association.