Understanding spatial patterns in population characteristics and the principal natal environments supporting riverine fish populations are important for fisheries management. Fin ray microchemistry was used to identify natal environment, and age estimates from sectioned fin rays were used to estimate growth and mortality rates for spotted bass, Micropterus punctulatus (Rafinesque), in a segment of the Ohio River (Smithland Pool) and three tributaries. Differences in water Sr:Ca and Ba:Ca among the Ohio River and tributaries were reflected in fin ray edge Sr:Ca and Ba:Ca. Fourteen percent of spotted bass ≥ age 2 captured in the Ohio River originated in tributaries, whereas 10% captured in tributaries originated in the Ohio River. Spotted bass in the Ohio River reached larger maximum size (L∞ = 448.7) than conspecifics in tributaries (L∞ = 324.4), although mortality rates were not different. Although 86% of spotted bass were collected in their inferred natal environment, small tributaries may be a supplemental source of recruitment for the spotted bass stock in Smithland Pool.