We evaluated reproductive isolation of Chinook salmon (Oncorhynchus tshawytscha) life history types that have been reintroduced to northern Idaho, USA. Analysis of 1003 samples at six microsatellite loci revealed strong reproductive isolation between ocean- and stream-type Chinook salmon (fall and summer spawn timing, respectively) within the Clearwater River sub-basin (F ST = 0.148, P < 0.00001). Very little evidence for gene flow among the two life history types was observed as assignment tests correctly assigned 99.6% of individuals in reference collections to either ocean- and steam-type Chinook salmon. Assignment of naturally reared juveniles indicated that both life history types were present with 24.1% stream-type and 75.9% ocean-type. Previous studies suggest high levels of divergence among the two life history types in natural populations, and our study verifies the persistence of reproductive isolation among types following colonization of habitat. Successful colonization of new habitat by (re)introduced species is likely influenced by diversity in life history types and this strategy has lead to naturally spawning populations in a variety of available habitats in the Clearwater River. As many populations of O. tshawytscha are listed as threatened or endangered under the U.S. Endangered Species Act, hope for recovery lies not only in effective management and habitat improvement, but adaptability of this species.