The Spotted Eagle Ray, Aetobatus narinari, is a semi-pelagic myliobatoid recognized as near threatened by the World Conservation Union. A decreasing population trend, K-selected life history, and primarily inshore, coastal habitat renders this species susceptible to over-exploitation by targeted fisheries, drift netting, and capture as bycatch. Recent molecular investigations and subsequent taxonomic recognition as a species complex have left a large deficiency in current knowledge of A. narinari and a complete absence of data concerning population structure and genetic health for the northwestern Atlantic Ocean. Our study is the first to examine fine-scale structure on a regional basis in the Gulf of Mexico and coastal Atlantic waters off the United States. Individuals sampled non-invasively from four sites, Sarasota, FL (n  =  143), the Everglades, FL (n  =  36), Ft. Pierce, FL (n  =  8), and the southeastern U.S. Atlantic coast (n  =  24), were genotyped across eight microsatellite loci. Standard tests for Hardy-Weinberg equilibrium, null alleles, linkage disequilibrium, allelic richness (mean  =  15.38), and gene diversity (0.701) were performed. Analyses of statistically significant patterns of geographic structure using STRUCTURE and AMOVA procedures yielded non-significant findings and support a single population model. Effective population size was estimated between 2,200 and 3,300 (LDNe and ONeSAMP, respectively). Assessments of structure and genetic health for the northwestern Atlantic are critical to furthering our understanding of viability of A. narinari.