This study aimed to investigate β-casein gene polymorphisms in Jersey cows in Japan. Blood samples were collected from 590 cows from eight Jersey farms in Okayama Prefecture, western Japan. Sequence analysis of exon 7 regions in chromosome 6 of the CSN2 gene revealed the genotype and allele frequencies of the β-casein variants. Considering that variant B belongs to the A1 group and variant I to the A2 group, plasma metabolite concentrations were compared among the A1A1, A1A2, and A2A2 group-based genotypes. The most frequent genotype was A2A2 (0.558), followed by A2B (0.190) and A2I (0.103). No variants of A3, F, G, H1, or H2 were found. The frequencies of group-based genotypes were A1A1 (0.032), A1A2 (0.303), and A2A2 (0.665). Although farm-to-farm differences were observed in the plasma concentrations of urea nitrogen, calcium, and phosphorus, no differences were found between the A1A1, A1A2, and A2A2 group-based genotypes; hence, the β-casein genotypes did not affect the metabolism of major nutrients. Owing to the high frequency of the A2 variant, Jersey cows can be considered an attractive breed for marker-assisted selection to create A2A2 herds.