In Lake Erie, lake whitefish Coregonus clupeaformis supported lucrative fisheries before populations were decimated by overfishing and water quality degradation. In recent years, there has been a renewed interest in lake whitefish and management of the fishery they support. Lake whitefish spawn on several reefs throughout Lake Erie, but the relative recruitment dynamics and contributions of spawning groups to the fishery are not well understood. Modern high-throughput sequencing approaches offer new opportunities to census population diversity and to identify subtle differences among closely related populations. We used high-throughput sequencing data to evaluate the genetic structure and diversity of lake whitefish collected opportunistically across broad spatial scales in Lake Erie. Using RAD-capture (Rapture), we sequenced and genotyped individuals (N = 88) from the west, central, and east basin of Lake Erie at 120,268 single nucleotide polymorphisms (SNPs). Lake whitefish from Niagara and Crib Reefs (west basin) diverged from the three collections. Interestingly, these were the only lake whitefish collected during the act of spawning (late November), and all other fish were collected pre-spawn (August-early November). These results suggest that some lake whitefish spawning reefs may be reproductively isolated, though definition of these groups into stocks will require more intentional sampling during the act of spawning.