Anthropogenic activities are placing increasing pressure on many species, particularly those that rely on more than one ecosystem. River herring (alewife, Alosa pseudoharengus and blueback herring, A. aestivalis collectively) are anadromous fishes that reproduce in rivers and streams of eastern North America and migrate to the western Atlantic Ocean. Here, we use data from single nucleotide polymorphisms (SNPs) to provide a comprehensive analysis of population structure for both species of river herring throughout their native ranges. We sampled river herring spawning runs in rivers from Newfoundland to Florida, examining a total of 108 locations, and genotyping over 8000 fish. We identified geographic population groupings (regional genetic groups) in each species, as well as significant genetic differentiation between most populations and rivers. Strong correlations between geographic and genetic distances (i.e., isolation by distance) were found range-wide for both species, although the patterns were less consistent at smaller spatial scales. River herring are caught as bycatch in fisheries and estimating stock proportions in mixed fishery samples is important for management. We assessed the utility of the SNP datasets as reference baselines for genetic stock identification. Results indicated high accuracy of individual assignment (76–95%) to designated regional genetic groups, and some individual populations, as well as highly accurate estimates of mixing proportions for both species. This study is the first to evaluate genetic structure across the entire geographic range of these species and provides an important foundation for conservation and management planning. The SNP reference datasets will facilitate continued multi-lateral monitoring of bycatch, as well as ecological investigation to provide information about ocean dispersal patterns of these species.