Intraspecific variations in specialized metabolites play a crucial role in the adaptive response to diverse environments. Two major subspecies, japonica and indica, are observed in Asian cultivated rice (Oryza sativa L.). Previously, we identified (3R)-β-tyrosine, a novel nonproteinogenic β-amino acid in plants, along with the enzyme tyrosine aminomutase (TAM1), required for β-tyrosine biosynthesis, in the japonica cultivar Nipponbare. Notably, TAM1 and β-tyrosine preferentially distributed in japonica cultivars compared with indica cultivars. Considering its phytotoxicity and antimicrobial activity, intraspecific variations in β-tyrosine may contribute to defensive potentials of japonica rice. Investigation of the evolutionary trajectory of TAM1 and β-tyrosine should enhance our understanding of evolution of rice defense. However, their distribution patterns in Oryza rufipogon, the direct ancestor of O. sativa, remain unclear. Therefore, in this study, we extensively examined TAM1 presence/absence and β-tyrosine contents involving 110 genetically and geographically diverse O. rufipogon and revealed that they are characteristically observed in the ancestral subpopulation of japonica rice, while being absent or slightly accumulated in other subpopulations. Thus, we conclude that TAM1 and β-tyrosine in japonica rice are likely derived from its ancestral subpopulation. Furthermore, the high and low TAM1 possession rates and β-tyrosine contents in japonica and indica rice, respectively, could be attributed to distribution patterns of TAM1 and β-tyrosine in their ancestral subpopulations. This study provides fundamental insights into evolution of rice defense.