Background <p><i>Sitophilus zeamais</i> (Coleoptera: Curculionidae) is a globally distributed pest of stored grain that is responsible for significant postharvest losses in grain-producing regions. In this study, morphological traits (body length, weight, and composition), enzyme characteristics, and reproductive isolation among geographically distinct <i>S. zeamais</i> populations in Japan were investigated.</p> Results <p>The results revealed population-specific differences in body length, weight, and composition. Nondenaturing polyacrylamide gel electrophoresis (Native–PAGE) analysis of cellulase enzymes revealed variations in the number and pattern of cellulase bands among populations, with a positive correlation observed between the number of bands and cellulase activity. Notably, individuals from the Sumika Technoservice Co., Ltd (STC) population exhibited uniform cellulase banding patterns, suggesting the fixation of enzyme traits due to regional characteristics or rearing history. Reproductive isolation experiments revealed reduced fertility in interpopulation crosses involving males from the National Agriculture and Food Research Organization (NARO) and rice store (RS) populations and females from the STC, Yoshinogari Historical Park (YHP), and rice farmer (RF) populations. Furthermore, low F₁ emergence within the NARO and STC populations suggested reduced reproductive ability, which may be linked to long-term artificial rearing. These findings indicated that population formation in <i>S. zeamais</i> could lead to both physiological differentiation and reproductive isolation.</p> Conclusions <p>The stored-grain pest <i>S. zeamais</i> underwent local evolution and reproductive isolation in Japan, especially in strains reared in research institutes.</p>