OBJECTIVE: To confirm the symptoms and signs for motor neuron disease (MND) in the Red Flag tool; to quantify the extent to which the key symptoms and signs are associated with MND; and to identify additional factors which may be helpful within the primary care setting in recognition of possible MND and triggering timely referral to neurology specialists. DESIGN: A nested case-control study. SETTING: 1292 UK general practices contributing to the QResearch primary care database, linked to hospital and mortality data. PARTICIPANTS: Baseline cohort included 16.8 million individuals aged 18 years and over without a diagnosis of MND at study entry and with more than 3 years of digitalised information available. The nested case-control data set comprised of 6437 cases of MND diagnosed between January 1998 and December 2019, matched by year of birth, gender, general practice and calendar year to 62 003 controls. MAIN OUTCOME MEASURES: Clinically recognised symptoms and signs of MND prior to diagnosis and symptoms and factors which are relevant in primary care setting. RESULTS: This study identified 17 signs and symptoms that were independently associated with MND diagnosis in a multivariable analysis. Of these, seven were new to the Red Flag tool: ataxia, dysphasia, weight loss, wheeze, hoarseness of voice, urinary incontinence and constipation. Among those from the Red Flag tool, dysarthria had the strongest association with subsequent MND (adjusted OR (aOR): 43.2 (95% CI 36.0 to 52.0)) followed by muscle fasciculations (aOR: 40.2 (95% CI 25.6 to 63.1)) and muscle wasting (aOR: 31.0 (95% CI 19.5 to 49.4)). Additionally, the associations between MND diagnosis and family history, dropped foot, focal weakness and sialorrhoea remained robust after controlling for confounders. Patients who reported symptoms indicative of damage to the lower brainstem and its connections were diagnosed sooner than those who presented with respiratory or cognitive signs. CONCLUSION: This is the first study that has identified, confirmed and quantified the association of key symptoms and signs with MND diagnosis. In addition to known factors, the study has identified the following new factors to be independently associated with MND prior to diagnosis: ataxia, dysphasia, wheeze and hoarseness of voice. These findings may be used to improve risk stratification and earlier detection of MND in primary care.