The genetic basis of sex determination is typically conserved within species if not within broader lineages. For example, within the stickleback family (Gasterosteidae), AmhY has been identified as a master sex-determination (MSD) gene in multiple species across two genera. By constrast, the existence of within-species variability in the genetic basis of sex determination is not frequently observed but provides an opportunity to understand the evolution and turnover of sex determination systems. In this study, we investigated the consistency with which AmhY is involved in sex determination across 610 individuals from five brook stickleback (Culaea inconstans) populations. We designed a PCR-restriction enzyme assay to identify the presence of AmhY in each individual and recorded sexual morphology in each individual in the field at the time of capture. We found that the genetic sex (presence/absence of AmhY) did not match the field-determined phenotypic sex in up to 27% of individuals within a population. This variation in the genetic basis of sex determination in brook stickleback suggests that the mechanism of sex determination in this species is likely more complex than thought when AmhY was first implicated and may still be evolving. Such within-species variation provides an opportunity to further investigate how and why transitions in sex-determination mechanisms occur.