Close relatives of the small genera Cualac (one species) and Floridichthys (two species) in the family Cyprinodontidae show sexual differences in chromosome number, with females having 2n = 48 and males having 2n = 47 with a large Y chromosome representing chromosomal fusion. However, karyotypic analysis of both sexes in F. carpio and C. tesselatus revealed the typical cyprinodontid karyotype, with 2n = 48, one normal-sized metacentric to submetacentric chromosome pair, and a graded series of 23 acrocentric or subtelocentric pairs. The phylogenetic pattern suggests that sexually dimorphic karyotypes have arisen independently on two separate occasions in a group that otherwise shows unusually high stability in chromosome morphology.