The Cherokee darter (Etheostoma scotti) is a Federally Threatened percid fish endemic to the Etowah River system, Georgia. Research was conducted on aspects of life history and genetic structure of this fish to aid in the protection and management of the species and its habitat. Through snorkel observations spawning behavior, spawning season, and preferred spawn habitat conditions were determined through two field seasons. Simultaneously, an analysis of population structure was made utilizing mtDNA with neighbor joining, maximum parsimony, and Bayesian analyses. Field observations found the habitat parameters required or preferred by Cherokee darters for spawning varied, with spawning occurring between the middle of March and early June. Genetic results show three distinct groups within the greater population of Cherokee darters in the Etowah River basin. These groups correspond with geographic breaks within the range of the species and fall out in the order of upper, middle and lower in terms of the Etowah drainage. These ESUs are supported, in part, by color variation in nuptial males between the ESUs.