Lewis blood group status is determined by two fucosyltransferase activities: those of FUT2-encoded fucosyltransferase (Se enzyme) and FUT3-encoded fucosyltransferase (Le enzyme). In Japanese populations, c.385A>T in FUT2 and a fusion gene between FUT2 and its pseudogene SEC1P are the cause of most Se enzyme-deficient alleles (Sew and sefus), and c.59T>G and c.314C>T in FUT3 are tag SNPs for almost all nonfunctional FUT3 alleles (le59, le59,508, le59,1067, and le202,314). In this study, we first conducted a single-probe fluorescence melting curve analysis (FMCA) to determine c.385A>T and sefus using a pair of primers that collectively amplify FUT2, sefus, and SEC1P. Then, to estimate Lewis blood group status, a triplex FMCA was performed with a c.385A>T and sefus assay system by adding primers and probes to detect c.59T>G and c.314C>T in FUT3. We also validated these methods by analyzing the genotypes of 96 selected Japanese people whose FUT2 and FUT3 genotypes were already determined. The single-probe FMCA was able to identify six genotype combinations: 385A/A, 385T/T, sefus/sefus, 385A/T, 385A/sefus, and 385T/sefus. In addition, the triplex FMCA successfully identified both FUT2 and FUT3 genotypes, although the resolutions of the analysis of c.385A>T and sefus were somewhat reduced compared to that of the analysis of FUT2 alone. The estimation of the secretor status and Lewis blood group status using the form of FMCA used in this study may be useful for large-scale association studies in Japanese populations.