Objective Traditional serological method for detecting the ABO blood group in humans are unsuitablefor macaques. Detecting the ABO blood group in macaques is important for transplantation and research. This study aimed toestablish a rapid and reliable nucleic acid-based method for identifying the ABO blood group in Macaca fascicularis andMacaca mulatta. Methods A new molecular method for ABO blood group detection was established, using competitiveallele-specific PCR (KASP) to identify the Macaca fascicularis and Macaca mulatta’ blood group by identifying the basesat 2 single nucleotide polymorphism (SNP) loci that are closely related to the blood-type functional gene. The bloodsamples identified via the KASP method were identified by Sanger sequencing by designing PCR primers around the twoSNP loci. Results Fifteen whole-blood nucleic acid samples from Macaca fascicularis and Macaca mulatta were determinedby TaqMan PCR and compared. The newly established KASP typing result were consistent with the original blood typeresult. To ensure the accuracy of the KASP method identification result, we used the newly established PCR method verifiedby Sanger sequencing, showing that the KASP method was consistent with the sequencing results. To verify the repeatabilityof the new KASP method, six blood samples with known blood groups were selected, and five replicates were taken perblood sample. The results showed that the method had excellent repeatability. In addition, 51 clinical samples were detectedusing the new KASP method, including 38 samples from cynomolgus monkeys and 13 from rhesus monkeys. The blood typeresults for two sites from the same sample were consistent, of which, 9 were type A (17. 6%), 19 were type B (37. 3%),23 were type AB (45. 1%), and none was type O. From the clinical samples with types A, B, and AB, five of each wereselected for PCR amplification and sequencing analysis, which confirmed that the sequences at the two SNP loci wereconsistent with those of the KASP typing. Conclusions The new detection method using KASP is highly accurate and canbe used to rapidly detect the ABO blood types in macaques. Compared with traditional serology and sequencing method, thismethod is simple, rapid, economical and reliable, and the results are easily interpreted. Furthermore, the samplerequirements are lower than those of traditional serological method, and fresh or long-stored whole blood, saliva or nucleic acid can be used.