The Red Shiner Cyprinella lutrensis is of increasing management interest as an invasive species that negatively impacts many native fishes throughout North America. Trojan sex chromosome (TSC)-carrying individuals could theoretically control invasive fish populations by skewing the sex ratio to 100% male. The efficacy of TSC-based control programs requires an understanding of a population's sex determination system, yet such information is lacking for Red Shiner. We used single-digest restriction site-associated DNA sequencing to discover sex-linked single-nucleotide polymorphisms (SNPs), and we conducted a series of breeding experiments to uncover the sex determination system. All candidate sex-linked SNPs that fit our selection criteria exhibited a pattern of male heterogamety. We developed two sex-identification (sex-ID) marker assays, XY_248 and XY_170, which showed phenotype–genotype concordance scores of 77.00% and 84.35%, respectively. These sex-ID markers exhibited relatively high phenotype–genotype concordance in females (XY_248 = 96.30%; XY_170 = 98.61%), which allowed for selective breeding of phenotypically feminized genetic males. We observed a 3:1 male : female sex ratio in spawns from feminized males crossed with wild-type males, indicative of a male heterogametic sex determination system (i.e., XY male/XX female). The discovery of a male heterogametic sex determination system, in combination with our two markers, increases the likelihood of developing an effective TSC eradication strategy for invasive Red Shiner populations.