It has been suggested that intramyocellular diglycerides may be associated with insulin resistance and thus may be linked to the pathophysiology of diabetes. We aimed to investigate intramyocellular diglyceride as well as triglyceride levels in diabetic subjects and to explore a possible association with glycemic control. The participants of the study were 30 obese subjects stratified according to the presence of diabetes into nondiabetic obese (n = 19) and diabetic obese (n = 11). Intramyocellular triglycerides and diglycerides were determined in biopsies from the vastus lateralis muscle under fasting conditions. Glycemic control and insulin resistance were assessed by an oral glucose tolerance test and the homeostatic model, respectively. Higher levels of intramyocellular triglycerides were observed in the diabetic obese group compared with the nondiabetic obese group (66.67 ± 23.75 vs 18.35 ± 4.42 nmol·mg⁻¹ dry tissue, respectively; P < .05). Diglyceride levels were not significantly different between the study groups (1.65 ± 0.27 vs 1.94 ± 0.65 nmol·mg⁻¹ dry tissue, respectively). Monounsaturated fatty acids represented the major constituent of intramyocellular triglycerides in both groups, whereas diglycerides contained mainly saturated fatty acids. A significant correlation was found between intramyocellular levels of triglycerides, but not diglycerides, and glycemic control, expressed as the area under the glucose curve (r = 0.417, P < .05). No correlations were found between intramyocellular levels of both lipid classes and insulin resistance. Our data support a relationship between glycemic control and intramyocellular triglycerides, but not diglycerides. The total flux of fatty acids toward esterification may be a much more important factor in the pathophysiology of diabetes.