Background: We developed an allele-specific polymerase chain reaction (AS-PCR) technique for Kidd blood group genotyping. Methods: Altogether, 340 blood samples from Thai blood donors at the National Blood Centre, Thai Red Cross Society, were tested with anti-Jka and anti-Jkb using the gel technique and the direct urea lysis test was used for screening Jk(a-b-) phenotype. For AS-PCR technique, different types of primers were used for JK*01 and JK*02 allele detections in known DNA controls. Results: Regarding JK*02 allele detection, the pseudopositve amplification products were found when using correctly matched forward primer and a single mismatch forward primer. Interestingly, one type of two mismatch pairing at the 3′ end of the forward primer can be used together with the newly designed reverse primer for Kidd blood group genotyping. It was found that the typing results in all samples obtained by serological techniques and newly developed AS-PCR technique were in agreement and this PCR technique also gave 100% concordance of results in 30 samples randomly tested twice and demonstrated reproducible results. Conclusion: This study shows that the in-house AS-PCR is simple, cost-effective, and convenient for Kidd blood group genotyping in routine laboratories, especially, in resolving serologic investigations. © 2012 Wiley Periodicals, Inc.