Although Agaricus bisporus mushroom is a popular mushroom consumed world-widely, the application of common bio-elements to verify its geographical origin remains highly limited. Therefore, this study aimed to verify whether the six cultivation regions in Korea of A. bisporus could be determined by the stable isotope composition analysis of bio-elements, which are unique and abundant in most living creatures. δ13C, δ15N, δ18O, and δ34S in A. bisporus were influenced by the region, cultivar, and the interactions between these two factors (P < 0.05). In particular, the effect of cultivation region was more significant to the isotope ratio profiles as compared to the mushroom cultivar effect. During the cultivation period of A. bisporus, the C, N, O, and S isotopic fractionation was observed between the mushroom and cultivation medium, note higher in the mushroom (P < 0.05). Two dimensional plot of δ15N, δ18O, or δ34S effectively distinguished the cultivation regions, Nonsan, Buyeo, Boryung, Daegu, and/or Gyeongju examined in this study. Further, these isotope ratio profiles measured in this study would be statistically analyzed with various chemometrics to provide isotope markers for the authenticity of geographical origin. Our preliminary case study improves our understanding of how the isotope composition of A. bisporus varies with respect to cultivation regions and cultivars. In conclusion, the analysis of stable isotope ratios is a suitable potential tool for discrimination between the cultivation origins of A. bisporus collected from Korea, with potential application to other countries after certain validation steps required.