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As fuel injection strategies in spark-ignition (SI) engines have been diversified, inhomogeneous mixing of the fuel-air mixture can occur to varying extents during mixture preparation. In this study, we analyzed the effect of inhomogeneous mixing on the knocking characteristics of iso-octane and air mixture under a standardized fuel testing condition for research octane number (RON), based on ASTM D2699. For this purpose, we assumed that both lean spots and rich spots existed in unburned gas during compression stroke and flame propagation and calculated the thermodynamic state of the spots by using an in-house multi-zone, zero-dimensional SI engine model. Then, the ignition delay was measured over the derived thermodynamic profiles by using rapid compression machine (RCM), and we calculated ξ, the ratio of sound speed to auto-ignition propagation speed, based on Zel’dovich and Bradley’s ξ − ε theory to estimate knock intensity.