Purpose Co a and Co b antigens of the Colton (CO) blood group system are implicated in acute and delayed hemolytic transfusion reactions (HTRs). Owing to the inadequate supply of specific antiserum, data on CO phenotypes remain limited. This study aimed to develop genotyping methods to predict Co a and Co b antigens and to estimate transfusion-induced alloimmunization risks in three Thai blood donor populations. Materials and Methods The study included 2451 blood samples from unrelated healthy Thai blood donors obtained from central, northern, and southern Thailand. DNA sequencing was used to determine the CO*A and CO*B alleles. In-house PCR with sequence-specific primers (PCR-SSP) and high-resolution melting curve (HRM) assays were performed and genotyping results were compared using DNA sequencing. CO*A and CO*B allele frequencies among Thais were determined using PCR-SSP and their frequencies were compared with other populations. The risks of Co a and Co b transfusion-induced alloimmunization among Thai donor populations were calculated. Results The validated genotyping results by PCR-SSP and HRM assays agreed with DNA sequencing. The CO*A / CO*A was the most common (100.0, 100.0, and 99.3%), followed by CO*A / CO*B (0.0, 0.0, and 0.7%) among central, northern and southern Thais. Homozygous CO*B / CO*B was not found. The CO*A and CO*B allele frequencies among central Thais significantly differed compared among southern Thais ( p < 0.01) but not among northern Thais. Those allele frequencies among Thais were similar to those of Taiwanese, Chinese and Malay-Malaysian populations but not to South Asian, Southeast Asian, Korean, Japanese, Filipino, French Basque, and Maltese populations ( p < 0.01). A higher risk of anti-Co b production rather than anti-Co a production was particularly noted in the southern Thai population. Conclusion This study constitutes the first to determine CO*A and CO*B genotypes using PCR-SSP and HRM assays among Thais and this finding would be beneficial in predicting alloimmunization risk and providing safe transfusions among Thais.