Obovaria olivaria is a species of freshwater mussel native to the Mississippi River and Laurentian Great Lakes-St. Lawrence River drainages of North America. This mussel has experienced population declines across large parts of its distribution and is imperiled in many jurisdictions. Obovaria olivaria uses the similarly imperiled Acipenser fulvescens (Lake Sturgeon) as a host for its glochidia. We employed mitochondrial DNA sequencing and Restriction-site Associated DNA sequencing (RAD-seq) to assess patterns of genetic diversity and population structure of O. olivaria from 19 collection locations including the St. Lawrence River drainage, the Great Lakes drainage, the Upper Mississippi River drainage, the Ohioan River drainage and the Mississippi Embayment. Heterozygosity was highest in Upper Mississippi and Great Lakes populations, followed by a reduction in diversity and relative effective population size in the St. Lawrence populations. Pairwise F_ST ranged from 0.00 to 0.20, and analyses of genetic structure revealed two major ancestral populations, one including all St. Lawrence River/Ottawa River sites and the other including remaining sites; however, significant admixture and isolation by river distance across the range were evident. The genetic diversity and structure of O. olivaria is consistent with the existing literature on Acipenser fulvescens and suggest that, although northern and southern O. olivaria populations are genetically distinct, genetic structure in O. olivaria is largely clinal rather than discrete across its range. Conservation and restoration efforts of O. olivaria should prioritize the maintenance and restoration of locations where O. olivaria remain, especially in northern rivers, and to ensure connectivity that will facilitate dispersal of Acipenser fulvescens and movement of encysted glochidia.