Background <p>Determining the most appropriate level of amputation in patients with diabetes mellitus has not been well established. The purpose of this study is to determine whether muscle biopsy reveals predictive information about the success rate of patients undergoing diabetic major lower limb amputation.</p> Methods <p>A prospective observational study was conducted among diabetic patients who underwent below-knee amputation. Skin-subcutaneous and muscle biopsy samples were obtained during the operation from 62 patients who undergo major limb amputation. Depending on the complications after surgery, patients were assigned into three groups: Group 1 consisted of patients with adequate wound healing without any complications; Group 2 included patients with prolonged wound healing requiring additional interventions like debridement; and Group 3 consisted of patients who underwent reamputation at a more proximal level. Biopsy samples of the groups were compared regarding degenerative cells, inflammatory cells, and the presence of infection.</p> Results <p>There was a significant difference between Groups 1 and 3 regarding the presence of abscess formation and infection (<i>p</i> &lt; 001). Comparison of Groups 1 and 3 revealed significant differences regarding inflammatory cell count, respectively (<i>p</i> &lt; 001). According to the results of the ROC analysis performed for histopathologic cellular evaluation, 15% for inflammatory cell ratio in muscle samples and 25% for degenerative cell ratio both in muscle and skin samples were determined as cut-off values.</p> Conclusions <p>The presence of increased degenerative cell count and infection in muscle biopsy areassociated with higher rates of reoperation. The present study revealed that preoperative muscle biopsy has predictive value in patients undergoing major limb amputation.</p> Level of evidence <p>Level II, Prospective observational study.</p>