Sympatric populations of Atlantic cod with distinct spawning times in winter and spring have been identified within the Gulf of Maine. A new western Gulf of Maine stock assessment unit in U.S. waters lumps winter and spring spawning populations into a single stock unit and future monitoring and assessment of their abundance will require mixed stock analysis. The goal of this study was to evaluate the utility of otolith-based stock identification techniques to discriminate spawning populations. Significant differences in otolith structure and microchemistry were identified between winter and spring spawners with the highest degree of classification accuracy based on both features. Mixed stock analysis applied to fishery samples of unknown origin indicated the contemporary (2015-2016) commercial fishery is dominated by winter spawners. Exploratory classification of historical fishery samples suggested a change over time with spring and winter spawners present in near equal proportion in the early 1990s and winter spawners dominating in the early 1980s. Otolith-based stock composition analysis has the potential to support monitoring and assessment of cod populations in the Gulf of Maine.