In the past decade, several large-scale mortality events have occurred in the winter at Atlantic salmon (Salmo salar) sea-cage sites in Eastern Canada and Iceland. In recent lab-based studies, we held post-smolt salmon at ~ 3o C, and ~ 30% of these fish developed symptoms similar to what has been described as ‘Winter Syndrome’ or ‘Winter Disease’ (WS/WD) in bream and yellow drum at cold temperatures. In these species, fatty liver disease (FLD) is associated with, and thought to be one of the major causes of, WS/WD. To definitively determine if these salmon developed FLD (which has not been reported previously), we measured hepatic lipid class and fatty acid levels, and the transcript expression of 34 key markers of FLD in fishes and non-alcoholic fatty liver disease (NAFLD) in mammals, in salmon characterized as ‘asymptomatic’ (healthy) vs. those exhibiting ‘early’ and ‘advanced’ symptoms of WS/WD. We also correlated our results with previously reported phenotypic characteristics associated with disease progression in these same individuals. Total lipid and triacylglyceride (TAG) levels increased by ~50% in fish with symptoms of FLD, while sterols and acetone-mobile polar lipids decreased by ~30%. Finally, multivariate analyses revealed that the 3 groups of fish were clearly different, with saa5 (acute phase protein) contributing the greatest to the difference between asymptomatic and symptomatic fish, and cyp7a1b (involved in cholesterol catabolism) contributing the most to the difference between fish with ‘early’ vs. ‘advanced’ symptoms. In summary, our results provide very strong evidence that prolonged cold exposure can trigger FLD in this important aquaculture species.