BackgroundGenetic testing in amyotrophic lateral sclerosis (ALS) is increasingly recommended for all patients, but real-world implementation in middle-income countries remains heterogeneous. Brazilian motor neuron disease genetics has largely been characterized through protocol-based cohorts focused on the founder VAPB p.Pro56Ser variant (amyotrophic lateral sclerosis type 8, ALS8). The broader genetic landscape beyond ALS8 in Brazilian clinical populations has not been described in a large multiregional cohort. We aimed to characterize this profile.MethodsWe retrospectively analyzed 1,911 patients with motor neuron disease followed at six SARAH Network rehabilitation hospitals across Brazil between 2007 and 2024. Genetic testing was clinically directed; ALS8 cases (n = 78) were characterized separately. We assessed characteristics of genetically tested versus untested patients, the temporal evolution of testing modalities, and the diagnostic yield by gene and by Byrne family history classification.ResultsOf 1,833 patients without ALS8, 318 (17.3%) underwent genetic testing, with selection bias toward younger age, higher educational attainment, and positive family history of ALS or frontotemporal dementia (all p < 0.001). Testing expanded markedly after 2020, particularly C9orf72 screening and next-generation sequencing panels. Panel/exome sequencing identified pathogenic or likely pathogenic variants in 13 of 66 patients (19.7%), consistent with international literature for mixed familial-sporadic cohorts. Across all modalities, 30 patients without ALS8 carried pathogenic or likely pathogenic variants in six genes: SOD1 (n = 15), C9orf72 (n = 11; 7.0% of 157 tested), and VCP, FUS, TBK1, and SETX (n = 1 each). ATXN2 intermediate-length CAG repeats were identified in 15 of 230 patients tested (6.5%), replicating the 6.3% previously reported in a Brazilian multicenter sample. Yield by Byrne classification followed a marked gradient (Definite: 89.7%, Probable: 24.0%, Possible: 15.4%, Sporadic: 4.9%; p < 0.001). The SOD1 spectrum included recurrent variants of likely Iberian/European origin.ConclusionThis 18-year cohort defines a real-world genetic profile of motor neuron disease beyond ALS8, with diagnostic yield comparable to the literature and a Byrne gradient supporting universal testing. Beyond family history, socioeconomic factors appeared to shape access to testing, reinforcing the importance of equitable genetic testing for diagnostic precision, family counseling, and access to gene-targeted therapy.