BACKGROUND: Evidence indicates that genetic variations in the GYP(B-A-B) hybrid genes are associated with protection against malaria. Therefore, this study aims to characterize the GYP(B-A-B) hybrid alleles among northern Thais, Burmese, and Karen with and without a previous history of malaria infection. METHODS: A total of 709 DNA samples were genotyped to identify GYP(B-A-B) hybrids using PCR-sequence specific primers (PCR-SSP) combined with Sanger sequencing. Additionally, some DNA samples (n = 243) were also tested with high-resolution melting (HRM) analysis. RESULTS: In our sampled populations, 14/87 (16.0%), 3/34 (8.8%), 0/16 (0%), and 1/18 (5.6%) of northern Thais, Burmese, Karen, and other minorities in Myanmar with a previous history of malaria infection, respectively, were identified with GYP(B-A-B) hybrid genes, whereas individuals without a history of malaria infection were 24/155 (15.5%), 5/183 (2.6%), and 4/216 (1.9%) in northern Thais, Burmese, and Karen, respectively. In the latter groups, DNA sequences showed that 17/155 (11.0%) northern Thais were GYP*Mur/GYPB heterozygotes and the other 6/155 (3.9%) were GYP*Thai/GYPB heterozygotes. The remaining one (0.6%) sample was a GYP*Mur/GYP*Mur homozygote. Among Burmese, 3/183 (1.6%) were GYP*Mur/GYPB heterozygotes and 1/183 (0.5%) was GYP*Thai/GYPB heterozygote. The remaining one (0.5%) sample being a GYP*Mur/GYP*Mur homozygote. Among Karen samples, all four were GYP*Mur/GYPB heterozygotes. CONCLUSION: Across all studied populations, GYP*Mur was the predominant allele, followed by GYP*Thai. In addition, genotyping results obtained by HRM were consistent with PCR-SSP combined with Sanger sequencing. A statistically non-significant association was noted for the glycophorin GYP(B-A-B) hybrids and malaria infection. Our findings provide insight into genetic variations of GYP(B-A-B) hybrid alleles among populations in the Thailand-Myanmar border area. This information could be used as a guideline to identify compatible blood products for transfusion and to prevent alloimmunization.