The objectives of this study were to 1) examine skeletal muscle fatty acid oxidation in individuals with varying degrees of adiposity and2) determine the relationship between skeletal muscle fatty acid oxidation and the accumulation of long-chain fatty acyl-CoAs. Muscle was obtained from normal-weight [n = 8; body mass index (BMI) 23.8 ± 0.58 kg/m²], overweight/obese (n = 8; BMI 30.2 ± 0.81 kg/m²), and extremely obese (n = 8; BMI 53.8 ± 3.5 kg/m²) females undergoing abdominal surgery. Skeletal muscle fatty acid oxidation was assessed in intact muscle strips. Long-chain fatty acyl-CoA concentrations were measured in a separate portion of the same muscle tissue in which fatty acid oxidation was determined. Palmitate oxidation was 58 and 83% lower in skeletal muscle from extremely obese (44.9 ± 5.2 nmol · g⁻¹ · h⁻¹) patients compared with normal-weight (71.0 ± 5.0 nmol · g⁻¹ · h⁻¹) and overweight/obese (82.2 ± 8.7 nmol · g⁻¹ · h⁻¹) patients, respectively. Palmitate oxidation was negatively (R = −0.44, P = 0.003) associated with BMI. Long-chain fatty acyl-CoA content was higher in both the overweight/obese and extremely obese patients compared with normal-weight patients, despite significantly lower fatty acid oxidation only in the extremely obese. No associations were observed between long-chain fatty acyl-CoA content and palmitate oxidation. These data suggest that there is a defect in skeletal muscle fatty acid oxidation with extreme obesity but not overweight/obesity and that the accumulation of intramyocellular long-chain fatty acyl-CoAs is not solely a result of reduced fatty acid oxidation.