The Alligator Gar Atractosteus spatula (AG) is a long-lived fish of growing management and conservation interest. Situated on the border of Texas and Oklahoma, Lake Texoma supports one of the last robust AG populations in Oklahoma; however, a genetic evaluation of this population is lacking. We genotyped AG individuals with 17 microsatellite loci, 7 of which also cross-amplified in three sympatric Lepisosteus species: the Longnose Gar L. osseus (LN), Shortnose Gar L. platostomus (SN), and Spotted Gar L. oculatus (SP). Bayesian assignment analyses conducted in STRUCTURE and NewHybrids confirmed that a field-identified hybrid was an F₁ AG × LN and identified five other individuals that were suspected backcrosses (three LN × SN; two SN × SP). Alligator Gar had the lowest observed heterozygosity (0.179) and the lowest allelic richness (1.682) among the nonhybrid individuals of the four gar species examined. We also examined the potential for population structure and differences in pairwise relatedness (r) between two areas where AG are commonly encountered within Lake Texoma: the Red River and Washita River arms. No population structure was detected using noninformative or location priors in STRUCTURE, and estimates of r produced by the TrioML estimator in COANCESTRY were not significantly different between arms (overall mean r = 0.199). Point estimates of effective population size ranging from 16.3 to 29.2 suggested that the AG population may be vulnerable to the effects of inbreeding depression and random genetic drift. Results provide a genetic status assessment of AG in Lake Texoma and a baseline for future management and conservation decisions within Lake Texoma and surrounding regions.