Chemical signatures in otoliths and muscle tissue of albacore (Thunnus alalunga) from two regions of the North Pacific were characterized to examine population structure. Regions included northern (offshore northwest United States and Canada, <40N) and southern (offshore southwest United States and Mexico, >40N) areas where albacore have demonstrated region-specific differences in movement and size at age. Juvenile (ages 14 years) albacore were collected from each region through recreational and commercial fisheries over a 5-year period (20092013). Three different otolith chemistry assays were performed, including whole otoliths (proxy for lifetime signature), otolith edge (recent signature), and otolith core (nursery signature). Whole otolith 13C and 18O showed similar 13C and enriched 18O (0.5) values for juvenile albacore collected in the southern region; classification success to the two regions ranged from 78 to 91% during each year (similar age classes). Otolith edge 13C and 18O showed similar results as whole otoliths, but no regional differences were found for otolith core chemistry. Incorporation of trace element concentrations from whole otoliths improved discrimination between regions to 90100% classification success during each year of the study, with significantly higher Ba:Ca, Mg:Ca, and Mn:Ca in albacore otoliths from the northern region. Albacore from the southern region also displayed enriched muscle 13C (1.21) and 15N (0.85) values relative to fish of the same size range from the northern region. Discrimination success between the two regions across all years using muscle 13C and 15N was 91%. Ultimately, results from this study suggest regional separation of albacore in the eastern North Pacific warranting region-specific vital rates in future modelling efforts.