Myocardial fat accumulation could occur in diseased hearts. The degree of heterogeneity is unknown because accurate assessment is difficult using conventional proton magnetic resonance spectroscopy techniques in a beating heart. The purpose of this study was to characterize the distribution of intramyocellular lipid content and to determine its association with disease characteristics. Proton magnetic resonance spectroscopy was performed on formalin-fixed slices of human hearts at various circumferential locations (N = 55). Twenty-nine percent of the hearts had the highest fat content measured in the septum, followed by posterior (27%), lateral (26%), and anterior (18%) wall. Age was significantly correlated with the mean fat percentages (r2 = 0.12, P = 0.007). Those who died from cardiovascular disease demonstrated significantly higher and more heterogeneous fat distribution than those who did not (1.62% ± 1.1% vs. 0.59% ± 0.4%, P = 0.002). In summary, septal fat content is representative of mean fat percentage. Fat content increases with age; fat distribution may be heterogeneous when associated with cardiovascular disease. Magn Reson Med, 2012. © 2012 Wiley Periodicals, Inc.