ObjectiveTo define the genetic landscape of amyotrophic lateral sclerosis (ALS) and assess the contribution of possible oligogenic inheritance, we aimed to comprehensively sequence 17 known ALS genes in 391 ALS patients from the United States.MethodsTargeted pooled‐sample sequencing was used to identify variants in 17 ALS genes. Fragment size analysis was used to define ATXN2 and C9ORF72 expansion sizes. Genotype–phenotype correlations were made with individual variants and total burden of variants. Rare variant associations for risk of ALS were investigated at both the single variant and gene level.ResultsA total of 64.3% of familial and 27.8% of sporadic subjects carried potentially pathogenic novel or rare coding variants identified by sequencing or an expanded repeat in C9ORF72 or ATXN2; 3.8% of subjects had variants in >1 ALS gene, and these individuals had disease onset 10 years earlier (p = 0.0046) than subjects with variants in a single gene. The number of potentially pathogenic coding variants did not influence disease duration or site of onset.InterpretationRare and potentially pathogenic variants in known ALS genes are present in >25% of apparently sporadic and 64% of familial patients, significantly higher than previous reports using less comprehensive sequencing approaches. A significant number of subjects carried variants in >1 gene, which influenced the age of symptom onset and supports oligogenic inheritance as relevant to disease pathogenesis. ANN NEUROL 2015;77:100–113