Intramuscular triglyceride (IMTG) content is linked to insulin resistance in humans. Similar to bioactive lipids, IMTG is localized in different compartments of skeletal muscle which may influence the impact on insulin sensitivity. Subsarcolemmal and intermyofibrillar localization of IMTG is known to alter the relationship to insulin sensitivity. However, more detailed subcellular localization of IMTG species has yet to be described. We evaluated subcellular localization of IMTG in lean (n=15), endurance trained athletes (n=16), obese (n=15), and type 2 diabetic (n=12) men and women. Muscle biopsies were fractionated into sarcolemmal, cytosolic, mitochondrial/endoplasmic reticulum, and nuclear compartments. IMTG were measured using LC/MS/MS, and insulin sensitivity using insulin clamps. Insulin sensitivity was significantly different between groups, with athletes>lean>obese>T2D (p<0.001). Most sarcolemmal IMTG species were significantly greater in obese and T2D compared to lean and athletes, but IMTG with only saturated acyl chains (C48:0, C50:0, C52:0) were significantly increased only in T2D. Sarcolemmal IMTG were inversely related to insulin sensitivity (p=0.009), and positively related to fasting insulin (p=0.004). Nuclear IMTG were significantly greater in T2D compared to lean and athletes. While there was not a significant difference in total cytosolic IMTG, saturated cytosolic IMTG species were significantly increased in T2D compared to lean and athletes, and saturated cytosolic IMTG were inversely correlated to insulin sensitivity (p=0.001). There were no significant differences between groups for IMTG concentration in the mitochondrial/ER compartment. Combined these data reveal previously unknown differences in subcellular IMTG localization, indicate the importance of sarcolemmal and nuclear IMTG to insulin sensitivity, and suggest saturated IMTG may be uniquely deleterious for muscle insulin sensitivity. Disclosure D.E. Kahn: None. S. Zarini: None. L. Perreault: Advisory Panel; Self; Novo Nordisk A/S, Sanofi. Speaker's Bureau; Self; AstraZeneca, Boehringer Ingelheim Pharmaceuticals, Inc., Janssen Pharmaceuticals, Inc., Merck & Co., Inc., Novo Nordisk A/S. S.A. Newsom: None. K.A. Harrison: None. B.C. Bergman: Advisory Panel; Spouse/Partner; AstraZeneca, Merck & Co., Inc., Novo Nordisk Inc. Funding National Institutes of Health (R01DK089170, RR-00036, P30DK048520)