Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL). We used microchip electrophoresis in combination with automatic image analysis to develop a novel high-throughput PCR-RFLP to type the gene sequences that encode BLV Tax 233. This method revealed that 233L-Tax is more prevalent than 233P-Tax in cattle in Japan. The proportion infected with BLV carrying the gene encoding 233L-Tax was significantly higher in Holstein cattle than in Japanese Black cattle. Holsteins infected with BLV encoding 233L-Tax had higher proviral loads than did Holsteins infected with BLV encoding 233P-Tax and Japanese Blacks infected with BLV encoding 233L-Tax or 233P-Tax. The novel method developed in this study will be a useful tool for identifying cattle harboring BLV with a higher risk of EBL and viral transmission.