Fusarium wilt, a soil-borne disease caused by the fungal pathogen Fusarium oxysporum f. sp. fragariae, threatens strawberry (Fragaria × ananassa) production worldwide. The spread of the pathogen, coupled with disruptive changes in soil fumigation practices, have greatly increased disease pressure and the importance of developing resistant cultivars. While resistant and susceptible cultivars have been reported, a limited number of germplasm accessions have been analyzed, and contradictory conclusions have been reached in earlier studies to elucidate the underlying genetic basis of resistance. Here, we report the discovery of Fw1, a dominant gene conferring resistance to Fusarium wilt in strawberry. The Fw1 locus was uncovered in a genome-wide association study of 565 historically and commercially important strawberry accessions genotyped with 14,408 SNP markers. Fourteen SNPs in linkage disequilibrium with Fw1 physically mapped to a 2.3 Mb segment on chromosome 2 in a diploid F. vesca reference genome. Fw1 and 11 tightly linked GWAS-significant SNPs mapped to linkage group 2C in octoploid segregating populations. The most significant SNP explained 85% of the phenotypic variability and predicted resistance in 97% of the accessions tested-broad-sense heritability was 0.96. Several disease resistance and defense-related gene homologs, including a small cluster of genes encoding nucleotide-binding leucine-rich-repeat proteins, were identified in the 0.7 Mb genomic segment predicted to harbor Fw1 DNA variants and candidate genes identified in the present study should facilitate the development of high-throughput genotyping assays for accurately predicting Fusarium wilt phenotypes and applying marker-assisted selection.