Numerous studies of population genetics in salmonids and other anadromous fishes have revealed that population structure is generally organized into geographic hierarchies (isolation by distance), but significant structure can exist in proximate populations due to varying selective pressures (isolation by adaptation). In Chignik Lakes, Alaska, anadromous Dolly Varden char (Salvelinus malma) spawn in nearly all accessible streams throughout the watershed, including those draining directly to an estuary, Chignik Lagoon, into larger rivers, and into lakes. Collections of Dolly Varden fry from 13 streams throughout the system revealed low levels of population structure among streams emptying into freshwater. However, much stronger genetic differentiation was detected between streams emptying into freshwater and streams flowing directly into estuarine environments. This fine-scale reproductive isolation without any physical barriers to migration is likely driven by differences in selection pressures across freshwater and estuarine environments. Estuary tributaries had fewer larger, older juveniles, suggesting an alternative life history of smolting and migration to the marine environment at a much smaller size than occurs in the other populations. Therefore, genetic data were consistent with a scenario where isolation by adaptation occurs between populations of Dolly Varden in the study system, and ecological data suggest that this isolation may partially be a result of a novel Dolly Varden life history of seawater tolerance at a smaller size than previously recognized.