Background: In this study, we identified a novel glycophorin variant (GP.MOT) in a Mia‐positive Japanese blood donor. The proband with this glycophorin variant was discovered by antigen screening of samples from 475,493 Japanese blood donors using monoclonal anti‐Mia. Study design and methods: Standard serological techniques and flow cytometry were performed. GP.MOT RBCs were examined by immunoblotting using anti‐GPA, anti‐MUT or anti‐Mur. Genome DNA was extracted from whole blood, and the GYPA/GYPB was analyzed by polymerase chain reactions and Sanger sequencing. Results: The MNS blood group of the proband was M + N + wS‐s + with the presence of other low‐frequency antigens including Mia, Mur, MUT, and KIPP. A 43‐kDa molecule, which is almost equivalent in size to glycophorin A (GPA), was identified by immunoblotting using monoclonal anti‐MUT and anti‐Mur. Sanger sequencing clearly indicated that the proband had two different GYPA*M alleles at SNP rs62334651 (GYPA*M232 + 55A and GYPA*M232 + 55G), as well as a GYP(B‐A) hybrid allele (GYP*MOT) with breakpoints located on pseudoexon 3 of GYPB from c.210 to c.219. Discussion We identified a hybrid glycophorin GP.MOT with the deduced unique amino acid sequence GPB (20–45)‐GPΨB (46–70)‐GPA (71–149), which has not been previously reported. [ABSTRACT FROM AUTHOR]