Population structure of Gray Snapper (Lutjanus griseus) in U.S. waters was assessed via analysis of allele and genotype distributions at 13 nuclear-encoded microsatellites and mitochondrial (mt)DNA haplotype distribution among samples from five localities in the northern Gulf of Mexico (Gulf) and one locality on the Atlantic coast of Florida. Exact tests of homogeneity over all microsatellites were significant for both allele (P = 0.004) and genotype (P = 0.020) distributions; homogeneity tests for mtDNA haplotype distributions were not significant (P = 0.940). Weak but significant divergence (Φ_CT = 0.007, P = 0.020) among localities (microsatellites) was indicated by spatial analysis of molecular variance (SAMOVA), where three distinct groups (one from the northwestern Gulf, one from the northcentral/northeastern Gulf, and one from the east coast of Florida) were inferred. Spatial autocorrelation analysis (microsatellites) revealed an isolation-by-distance effect among samples from the northern Gulf. Levels of genetic variation in both microsatellites and mtDNA were low as compared to other lutjanids in U.S. waters, and Bayesian analysis of genetic demography revealed a two to three order-of-magnitude decrease in effective population size of Gray Snapper over the past 5,300 or so years (0.05 quartile of 81 years). The evidence of genetically distinct stocks and the decline in effective population size have implications for management of Gray Snapper resources in U.S. waters.