We separated an Aw10 allele by allele-specific sequencing in two Aw10/B101 samples that had the AweakB phenotype. Two samples with the A102/B101 genotype were also tested as a control. The reverse primers using position 930 at exon7 were designed for allele-specific sequencing. The differential positions were a total of 52 points for distinguishing the A-allele from the B-allele. Although overlaps with another haplotype allele that showed a minor chromatographic peak were observed in almost all the points, the specific allele-separation rate was 100% (52/52) by assessing the dominance in the chromatographic peak height. Based on the separation rate in the two cases with Aw10/B101 and the two AB controls, allele-specific sequencing is a convenient and reliable method for the separating the A-allele and B-allele in a clinical laboratory