Evaluating genetic diversity of seagrasses provides insight into reproductive mode and adaptation potential, and is therefore integral to broader conservation strategies for coastal ecosystems. In this study, we assessed genetic diversity, population structure and gene flow in an opportunistic seagrass, Syringodium filiforme, in the Florida Keys and subtropical Atlantic region. We used microsatellite markers to analyze 20 populations throughout the Florida Keys, South Florida, Bermuda and the Bahamas primarily to understand how genetic diversity of S. filiforme partitions across the Florida Keys archipelago. We found low allelic diversity within populations, detecting 35-106 alleles across all populations, and in some instances moderately high clonal diversity (R = 0.04-0.62). There was significant genetic differentiation between Atlantic and Gulf of Mexico (Gulf) populations (FST = 0.109 ± 0.027, p-value = 0.001) and evidence of population structure based on cluster assignment, dividing the region into two major genetic demes. We observed asymmetric patterns in gene flow, with a few instances in which there was higher than expected gene flow from Atlantic to Gulf populations. In South Florida, clustering into Gulf and Atlantic groups indicate dispersal in S. filiforme may be limited by historical or contemporary geographic and hydrologic barriers, though genetic admixture between populations suggests exchange may occur between narrow channels in the Florida Keys, or has occurred through other mechanisms in recent evolutionary history, maintaining regional connectivity. The variable genotypic diversity, low genetic diversity and evidence of population structure observed in populations of S. filiforme resemble the population genetics expected for a colonizer species.