Certain endocrine-active toxicants have been reported to completely sex reverse both male and female individuals in amphibian, avian, fish, invertebrate, and reptile species, resulting in a phenotype indistinguishable from unaffected individuals. Detection of low-level sex reversal often requires large numbers of organisms to achieve the necessary statistical power, especially in those species with predominantly genetic sex determination and cryptic/homomorphic sex chromosomes. Here we describe a method for determining the genetic sex in the commonly used ecotoxicological model, the fathead minnow (Pimephales promelas). Analysis of amplified fragment length polymorphisms (AFLP) in a spawn of minnows resulted in detection of 10 sex-linked AFLPs, which were isolated and sequenced. No recombination events were observed with any sex-linked AFLP in the animals examined (n=112). A polymerase chain reaction (PCR) method was then developed that determined the presence of one of these sex-linked polymorphisms for utilization in routine toxicological testing. Analyses of additional spawns from our in-house culture indicate that fathead minnows utilize a XY sex determination strategy and confirm that these markers can be used to genotype sex; however, this method is currently limited to use in laboratory studies in which breeders possess a defined genetic makeup. The genotyping method described herein can be incorporated into endocrine toxicity assays that examine the effects of chemicals on gonad differentiation.