Unionid mussels are a highly imperiled macroinvertebrate group that have received substantial conservation attention, particularly with genomic survey tools in recent decades. Glebula rotundata, Round Pearlshell, is a unionid endemic to rivers, bayous, and estuaries of the Gulf Coastal Plain of North America. It is unusual among unionids for its tolerance of brackish water, which allows it to occupy estuarine habitats in coastal river drainages; yet no studies characterizing genetic variation across the species’ native range have been published. Thus, we conducted a range-wide population genomic analysis to investigate patterns of diversity, differentiation, and structure in G. rotundata. We applied ddRAD-seq procedures to genotype 242 G. rotundata individuals from 15 Gulf Coast tributaries, generating a dataset of 37,143 SNPs. Summaries of genetic diversity and differentiation revealed substantial divergence among populations and likely signs of isolation by distance. Strong clustering and phylogenetic structuring supported the distinction of three primary regions, correlated with biogeographic provinces of the Western, Central, and Eastern Gulf. Populations of the Eastern Gulf were particularly differentiated, reflecting historical isolation associated with sea level and river confluence changes during the Pleistocene era. These results indicate that G. rotundata populations are highly structured, with limited connectivity among drainages due to historical vicariance and dispersal mode. This may pose significant risks to long-term viability, warranting concern for G. rotundata as climate-driven coastal pressures like sea level rise and saltwater intrusion intensify. To mitigate some of these effects, managers should prioritize populations with unique genetic diversity and removal or mitigation of barriers (e.g., dams, saltwater barriers) that block natural upriver dispersal response of bivalves. Future research will be needed to quantify if localized adaptations (e.g., increased salinity tolerance) exist and to project the extent of damage to habitats and populations expected under future climate scenarios.