Anti-PP1Pk is produced by p individuals without prior red blood cell alloimmunization. This antibody can react over a wide thermal amplitude, has the potential to bind complement, and has caused hemolytic transfusion reaction, hemolytic disease of the fetus and newborn, and a high rate of spontaneous abortions. This report of two cases describes the genetic basis of p phenotype underlying anti-PP1Pk production and the development of a semi-nested polymerase chain reaction (PCR) assay for screening this observed mutation among Thai blood donors. Antibody detection and confirmation were examined by serologic testing. Genomic DNA was extracted from two Thai Burmese descendants with the p phenotype and a history of spontaneous abortions caused by anti-PP1Pk; the entire coding region of the A4GALT gene of each was sequenced and analyzed. Additionally, a semi-nested PCR assay of the observed mutation was developed and used for screening the genomic DNA of 1502 Thai blood donors. Anti-PP1Pk was identified and the p phenotype was confirmed in the two Thai individuals of Burmese descent. A single-base duplication (c.201dupC in exon 3) in the A4GALT gene was detected in both p patients. The duplication is consistent with the A4GALT*01N.21 allele associated with the p phenotype and anti-PP1Pk production. A semi-nested PCR assay was developed and subsequently used for mass screening for this mutation. The mutation was not found among the 1502 Thai blood donors tested with this newly developed assay.