Fusarium wilt race 1, caused by Fusarium oxysporum f. sp. lactucae, is one of the main diseases of lettuce in tropical and subtropical regions, including under summer conditions in Japan. Resistant lettuce cultivars have shown symptoms on occasion, which have led breeding programs to focus on the improvement of resistance phenotypes to race 1. Disease assays using Fusarium wilt race 1 were performed, and the lettuce cultivar ‘VI185’ was categorized as highly resistant, which was validated by subsequent population analyses. F2 individuals obtained from a cross between ‘VI185’ (highly resistant) and ‘ShinanoGreen’ (partially resistant) were analyzed, and an F3 population was subjected to an infection assay using the race 1 pathogen. ddRAD-seq analysis revealed two significant QTLs on LG7 (qFOL7.1) and LG8 (qFOL8.1) that accounted for the race 1 resistance phenotype. Furthermore, the loci that conferred elevated resistance originated from ‘VI185’ at both QTLs. Our results provide a foundation for understanding the QTLs for resistance to Fusarium wilt race 1 and provide evidence that genetic diversity is available for enhancing the current resistance in existing lettuce cultivars.