Diverse freshwater lacustrine fishes enter tributaries to spawn, but resident riverine members may also occupy these same tributaries. While mark-recapture and biotelemetry studies suggest reproductive isolation between such populations, the assertion has rarely been tested genetically. To address this question, Micropterus dolomieu (Smallmouth Bass) from the southern shoreline of Lake Erie were compared genetically to bass in adjacent tributaries. Results from mitochondrial DNA sequences support the hypothesis that lacustrine and riverine populations segregate. Furthermore, divergences among tributary populations were often as large as those divergences between lacustrine and riverine bass, suggesting that each river population may become genetically distinct.