The phospholipid composition of membranes can influence the physiological functioning of the cell or subcellular organelle. This association has been previously demonstrated in skeletal muscle, where cellular or subcellular membrane, specifically mitochondria, phospholipid composition is linked to muscle function. However, these observations are based on whole mixed skeletal muscle analysis, with little information on skeletal muscles of differing fiber-type compositions. These past approaches that used mixed muscle may have misidentified outcomes or masked differences. Thus, the purpose of this study was to compare the phospholipid fatty acid composition of subsarcolemmal (SS) mitochondria isolated from slow-twitch postural (soleus), fast-twitch highly oxidative glycolytic locomotory (red gastrocnemius), and fast-twitch oxidative glycolytic locomotory (plantaris) skeletal muscles. The main findings of the study demonstrated unique differences between SS mitochondrial membranes from postural soleus compared to the other locomotory skeletal muscles examined, specifically lower percentage mole fraction of phosphatidylcholine (PC) and significantly higher percentage mole fraction of saturated fatty acids (SFA) and lower n6 polyunsaturated fatty acids (PUFA), resulting in a lower unsaturation index. We also found that although there was no difference in the percentage mole fraction of cardiolipin (CL) between skeletal muscle types examined, CL of soleus mitochondrial membranes were approximately twofold more SFA and approximately two-thirds less PUFA, resulting in a 20–30% lower unsaturation and peroxidation indices. Thus, the results of this study indicate unique membrane lipid composition of mitochondria isolated from different skeletal muscle types, a potential consequence of their respective duty cycles.