Transthyretin amyloidosis (ATTR amyloidosis) is a rare systemic disorder characterized by the extracellular deposition of amyloid fibrils, which can affect multiple tissues. Lumbar spinal stenosis (LSS), a condition involving narrowing of the lumbar spinal canal, has been frequently associated with amyloid deposition in the ligamentum flavum (LF). This study aimed to evaluate the prevalence of ATTR deposits in LF samples obtained from patients undergoing LSS surgery at two Italian centers. A total of 37 patients were included, with LF thickness measured via pre-operative MRI scans. Amyloid deposits were detected in 27% of patients, all confirmed as ATTR by immunohistochemistry. DNA analysis revealed no pathogenic mutations in the TTR gene, suggesting that the detected amyloid fibrils originated from the wild-type protein. LF thickness values were consistent with those reported in literature, supporting LF thickening as a potential marker of amyloid deposition. These findings contribute to the understanding of ATTR involvement in LSS and highlight the need for further research to explore the pathophysiological mechanisms and clinical significance of amyloid deposits in the LF.