Resistance to multiple races of Fusarium wilt is considered a critical trait for lettuce (Lactuca sativa L.) cultivars, as it is directly related to grower profitability in Japan. We analyzed F2 individuals obtained from a cross between ‘VI185’ (resistance to race 2) and ‘ShinanoGreen’ (susceptible to race 2), and applied a bioassay to the F3 population using the race 2 pathogen. ddRAD-seq analysis showed that a single semi-dominant locus on LG1 (qFOL1.2) controls resistance, and that the genotype of a RAD-marker designated as LG1_v8_117.181Mbp showed complete co-segregation with the resistance phenotype based on the F2 population. Fine mapping by PCR-based markers further revealed that qFOL1.2 were located in the position of 116.468-117.974Mbp. The 42 cultivars were evaluated for the genotypes and phenotypes using the PCR-based makers designed at this region. As a result of genotyping, all of the susceptible cultivars had the same genotype in this region, but the resistant cultivars had the diversity in the genotype. Notably, the genotype of the PCR-based marker designated as LG1_v8_116.506Mbp was consistent across the 25 resistant cultivars. Thus, we reasoned that LG1_v8_116.506Mbp was a broadly useful marker for selection of race 2-resistance. Our results provide additional breeding technique for resistance to race 2, and can accelerate pyramiding of resistance loci to multiple races of fusarium wilt.