The purpose of this study was to quantify the content and localization of fat in Atlantic mackerel ( N = 17) in order to assess fish quality and understand the bioenergetics of different life stages for this species. Measurement of total fat content using chemical shift based water-fat separation (MR) imaging was performed both when the fish was most starved (June) and well fed (September). The results were compared to triglyceride content measured by gas chromatography in the same fish. For starved fish a fat content of 40 ± 23 mg/g tissue was estimated by MRI compared to 39 ± 16 by GC analysis ( p = 0.732). For well fed fish, however, there were no agreements between the two techniques (447 ± 101 by MRI; 212 ± 89 by GC; p = 0.032). This could be due to the presence of non-triglyceride lipids, and the two approaches different sensitivity to these lipids. The results demonstrate that chemical shift based MR imaging is a powerful method for visualizing the seasonal variations in fat distribution, allowing storage fat to be distinguished from volatile fat based on their differences in hydrolytic stages. These findings underpin the potential of this imaging technique to obtain quantitative, accurate and non-invasive measures of tissue fat content.