Neurofilament levels are elevated in many neurodegenerative diseases and have shown promise as diagnostic and prognostic biomarkers in Amyotrophic Lateral Sclerosis (ALS), the most common form of Motor Neuron Disease (MND). This study assesses serum neurofilament light (NFL) and neurofilament heavy (NFH) chain concentrations in patients with ALS, other variants of motor neuron disease such as Progressive Muscular Atrophy (PMA) and Primary Lateral Sclerosis (PLS), and a range of other neurological diseases. It aims to evaluate the use of NFL and NFH to differentiate these conditions and for the prognosis of MND disease progression. NFL and NFH levels were quantified using electrochemiluminescence immunoassays (ECLIA). Both were elevated in 47 patients with MND compared to 34 patients with other neurological diseases and 33 healthy controls. NFL was able to differentiate patients with MND from the other groups with a Receiver Operating Characteristic (ROC) curve area under the curve (AUC) of 0.90 (p< 0.001). NFL correlated with the rate of disease progression in MND (rho 0.758,p< 0.001) and with the ALS Functional Rating Scale (rho −0.335,p= 0.021). NFL levels were higher in patients with ALS compared to both PMA (p= 0.032) and PLS (p= 0.012) and were able to distinguish ALS from both PMA and PLS with a ROC curve AUC of 0.767 (p= 0.005). These findings support the use of serum NFL to help diagnose and differentiate types of MND, in addition to providing prognostic information to patients and their families.