Analysis of population genetic relationships reveals the signatures of current processes such as spawning behaviour and migration, as well as those of historical events including vicariance and climate change. This study examines these signatures through testing broad- to fine-scale genetic patterns among smallmouth bass Micropterus dolomieu spawning populations across their native Great Lakes range and outgroup areas, with fine-scale concentration in Lake Erie. Our primary hypotheses include whether genetic patterns result from behavioural and/or geographical isolation, specifically: (i) Are spawning groups in interconnected waterways genetically separable? (ii) What is the degree of isolation across and among lakes, basins, and tributaries? (iii) Do genetic divergences correspond to geographical distances? and (iv) Are historical colonization patterns from glacial refugia retained? Variation at eight nuclear microsatellite DNA loci are analysed for 666 smallmouth bass from 28 locations, including 425 individuals in Lake Erie; as well as Lakes Superior, Huron, and Ontario, and outgroups from the Mississippi, Ohio, St. Lawrence, and Hudson River drainages. Results reveal marked genetic differences among lake and river populations, as well as surprisingly high divergences among closely spaced riverine sites. Results do not fit an isolation-by-geographical-distance prediction for fine-scale genetic patterns, but show weak correspondence across large geographical scales. Genetic relationships thus are consistent with hypotheses regarding divergent origins through vicariance in glacial refugia, followed by colonization pathways establishing modern-day Great Lakes populations, and maintenance through behavioural site fidelity. Conservation management practices thus should preserve genetic identity and unique characters among smallmouth bass populations.