We assess the potential of using otolith chemistry to differentiate quillback rockfish (Sebastes maliger) within Puget Sound, Washington, where two distinct population segments (DPS) have been identified. Using opportunistic collections (1993–2003) of quillback rockfish (n=77; age range of 2–65 yrs.) we first sought to determine whether fish from different sites and regions could be differentiated based on the trace elemental concentrations at the edge of their otoliths (i.e., the chemical record of the fish’s recent history). Results of our quadratic discriminant function analysis (QDFA) indicated significant spatial variability for fish collected at relatively large (regions) and small (sites) spatial scales. Specifically, fish collected from regions in 2002 (San Juan Islands and southern Puget Sound) and 2003 (eastern and western Strait of Juan de Fuca) were correctly classified with 100% and 65% accuracy (based on jack-knife classification), respectively, while fish collected from sites in 1998 (Mukilteo and Foulweather) were classified with 100% accuracy. We also investigated whether we could differentiate fish that were collected from different DPS and regions by using elemental concentrations from their whole otolith (which represents environmental information over the lifetime of a fish). Results from the QDFAs indicated relatively high classification success (80%) when comparing fish collected from either different DPS (i.e., Northern Puget Sound and Puget Sound Proper DPS) or regions (i.e., western and eastern Strait of Juan de Fuca). Findings from this study highlight the value of otolith chemistry in the study of population structure of quillback rockfish in Puget Sound.