The aim of this study was to investigate beta-casein polymorphism among 320 Japanese cows sampled from eight dairy farms. We used a newly-developed genotyping method that involved collecting DNA from hairs and a Cycleave polymerase chain reaction (PCR) assay to detect the A1, A2, and B variants. Results revealed the presence of five genotypes (A1A1, A2A2, A1A2, A1B, and A2B). We found that the most common genotype was A2A2 (0.42), followed by A1A2 (0.39) and A1A1 (0.11). The A1B and A2B genotypes were less frequent (<0.05). The frequencies of alleles A1, A2, and B were calculated to be 0.32, 0.64, and 0.04, respectively. Our study is the first to show the current status of beta-casein polymorphisms in Japanese dairy farms. Given the adverse effects of A1 beta-casein on human health, attempts have been made to develop herds consisting solely of A2A2 cows. Our study provides a reference for improving cow populations in Japanese dairy farms. The Cycleave PCR-based assay we developed here can be used for rapid and reliable genotyping of bovine beta-casein.