Few studies have reported on the fine-scale population genetics of batoid species in the Atlantic basin. Here, we investigate the genetic diversity and population structure of the spotted eagle ray, Aetobatus narinari, sampled in the northeastern and southwestern parts of the Gulf of Mexico and in the northwestern Caribbean Sea. Samples were collected from 286 individuals sampled across 3 geographic localities. Estimates of divergence based on the mitochondrial cytochrome b gene and 10 nuclear microsatellite loci reveal weak but significant genetic structure among A. narinari populations in this region. Analysis of molecular variance estimates based on both marker types indicate significant differentiation between Florida and Mexico populations, while comparisons with Cuba suggest high levels of gene flow with rays from both Mexico and Florida. Conflicting results were found from the different marker types when sexes were analyzed separately underscoring the importance of applying multiple marker types when making inferences about population structure and sex-biased dispersal. Results from Bayesian clustering analyses suggest rays may be migrating south out of the Gulf of Mexico and into the northwestern Caribbean Sea. Given the impacts of fisheries on this species, coupled with the lack of population genetic data available, these findings offer valuable information to aid with conservation management strategies.