Abstract Phosphorylated TDP-43 (pTDP-43) aggregates in the cytoplasm of motor neurons and neuroglia in the brain are one of the pathological hallmarks of amyotrophic lateral sclerosis. Although the axons exceed the total volume of motor neuron soma by several orders of magnitude, systematic studies investigating the presence and distribution of pTDP-43 aggregates within motor nerves are still lacking. The aim of this study is to define the TDP-43/pTDP-43 pathology in diagnostic motor nerve biopsies performed on a large cohort of patients presenting with a lower motor neuron syndrome and to assess whether this might be a discriminating tissue biomarker for amyotrophic lateral sclerosis and non-amyotrophic lateral sclerosis cases. We retrospectively evaluated 102 lower motor neuron syndrome patients referred to our centre for a diagnostic motor nerve biopsy. Histopathological criteria of motor neuron disease and motor neuropathy were applied by two independent evaluators, who were blind to clinical data. TDP-43 and pTDP-43 were evaluated by immunohistochemistry, and results compared to final clinical diagnosis. We detected significant differences between amyotrophic lateral sclerosis and non-amyotrophic lateral sclerosis cases in pTDP-43 expression in myelinated fibres: axonal accumulation was detected in 98.2% of patients with amyotrophic lateral sclerosis versus 30.4% of non-amyotrophic lateral sclerosis samples (P < 0.0001), while concomitant positive staining in Schwan cell cytoplasm was found in 70.2% of patients with amyotrophic lateral sclerosis versus 17.4% of patients who did not have amyotrophic lateral sclerosis (P < 0.001). Importantly, we were also able to detect pTDP-43 aggregates in amyotrophic lateral sclerosis cases displaying normal features at standard histopathological analysis. Our findings demonstrated that a specific pTDP-43 signature is present in the peripheral nervous system of patients with amyotrophic lateral sclerosis, and could be exploited as a specific, accessible tissue biomarker. The detection of pTDP-43 aggregates within motor nerves of living patients with amyotrophic lateral sclerosis, occurring before axonal degeneration, suggests that this is an early event that may contribute to amyotrophic lateral sclerosis pathogenesis.