Local adaptation is a dynamic process driven by selection that can vary both in space and time. One important temporal adaptation for migratory animals is the time at which individuals return to breeding sites. Chinook salmon (Oncorhynchus tshawytscha) are excellent subjects for studying the genetic basis of temporal adaptation because their high seasonal homing fidelity promotes reproductive isolation leading to the formation of local populations across diverse environments. We tested for adaptive genetic differentiation between seasonal runs of Chinook salmon using two candidate loci; the circadian rhythm gene, OtsClock1b, and Ots515NWFSC, a microsatellite locus showing sequence identity to three salmonid genes central to reproductive development. We found significant evidence for two genetically distinct migratory runs in the Feather River, California (OtsClock1b: FST = 0.042, P = 0.02; Ots515NWFSC: FST = 0.058, P = 0.003). In contrast, the fall and threatened spring runs are genetically homogenous based on neutral microsatellite data (FST = –0.0002). Similarly, two temporally divergent migratory runs of Chinook salmon from New Zealand are genetically differentiated based on polymorphisms in the candidate loci (OtsClock1b: FST = 0.083, P-value = 0.001; Ots515NWFSC: FST = 0.095, P-value = 0.000). We used an individual-based assignment method to confirm that these recently diverged populations originated from a single source in California. Tests for selective neutrality indicate that OtsClock1b and Ots515NWFSC exhibit substantial departures from neutral expectations in both systems. The large FST estimates could therefore be the result of directional selection. Evidence presented here suggests that OtsClock1b and Ots515NWFSC may influence migration and spawning timing of Chinook salmon in these river systems.