ABO types obtained from evidentiary samples have been used effectively to obtain the initial information leading to the apprehension of culprits in Japanese criminal investigations. A simple ABO genotyping method using multiplex sequence-specific PCR and capillary electrophoresis was developed as a supplement to serological ABO typing. Limitations in predicting a phenotype based on genotype were evaluated using 1134 randomly selected Japanese peripheral blood samples. A concordance rate of 99.82% (1132/1134 samples) was found between genotypes and phenotypes defined as Groups A, B, AB, and O. Sequencing analysis revealed that one discrepant sample contained an O allele having a previously unreported point mutation at the primer binding site in exon 6, and another discrepant sample contained an O allele lacking the guanine deletion at nt 261 (the O301 allele). Therefore, the existence of such alleles must be given some consideration when predicting phenotype based on genotype.