Skeletal muscle lipid accumulation is associated with several chronic metabolic disorders, including obesity, insulin resistance (IR) and type 2 diabetes. The aim of this study is to evaluate whether static imaging time-of-flight-secondary-ion mass spectrometry (TOF-SIMS) equipped with a Bismuth-cluster ion source can be used for studying skeletal muscle lipid accumulation associated with obesity. Mouse gastrocnemius muscle tissues in 10-week-old obese ob/ob (n = 8) and lean wild-type C57/BL6 (n = 6) mice were analyzed by TOF-SIMS. Our results showed that signal intensities of fatty acids (FAs) and diacylglycerols (DAGs) were significantly increased in skeletal muscle of the obese ob/ob mice as compared to the lean wild-type mice. These differences were revealed through a global analytical approach, principal component analysis (PCA) of TOF-SIMS spectra, and ion-specific TOF-SIMS images. Region-of-interest (ROI) analysis showed that FA signal intensities within the muscle cell were significantly increased in ob/ob mice. Moreover, analysis of the ratio between different FA peaks revealed changes in monounsaturated FAs (MUFAs) and polyunsaturated FAs (PUFAs), which is in agreement with previous reports on obesity. These changes in FA composition were also reflected in the ratio of different DAGs or phosphatidylcholines (PCs) that contain different FA residues. Imaging TOF-SIMS together with PCA of TOF-SIMS spectra is a promising tool for studying skeletal muscle lipid accumulation associated with obesity.