Candy Darters (Etheostoma osburni) and Variegate Darters (E. variatum) are both native to West Virginia and Virginia. The geographic ranges of these two species were historically separated by Kanawha Falls, a natural barrier to fish dispersal located at Glen Ferris, WV. In the early 1980s, Variegate Darters or putative hybrids (E. osburni × E. variatum) were first collected at locations upstream of Kanawha Falls, and have since undergone range expansion. Hybridization with the Variegate Darter was one of the threats that led to the Candy Darter being proposed for listing under the U.S. Endangered Species Act in 2017. Genetic and morphologic data were examined for individuals from the New, Gauley, and Greenbrier river drainages. Individuals were genotyped using a suite of 5 diagnostic microsatellite loci to investigate potential hybridization. Widespread hybridization was found throughout populations of Candy Darters, with the geographic range of hybridization expanding from 2004 to 2014. A hybrid zone was observed, with the highest levels of Variegate Darter introgression representing the kernel within this zone and the locations of first-generation (F1) hybrids at the periphery. F1 hybrids were morphologically intermediate within and across characters for parental species. Introgressive hybridization threatens the genetic integrity of the Candy Darter, and may lead to population extirpation or extinction.