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Nonpathogenic surrogate microorganisms, with a similar or slightly higher thermal resistance of the target pathogens, are usually recommended for validating practical pasteurization processes. The aim of this study was to explore a surrogate microorganism in wheat products by comparing the thermal resistance of three common bacteria in wheat kernels and flour. The most heat-resistant <i>Enterococcus faecium</i> NRRL-2356 rather than <i>Salmonella</i> cocktail and <i>Escherichia coli</i> ATCC 25922 was determined when heating at different temperature–time combinations at a fixed heating rate of 5 °C/min in a heating block system. The most heat-resistant pathogen was selected to investigate the influences of physical structures of food matrices. The results indicated that the heat resistance of <i>E. faecium</i> was influenced by physical structures of food matrices and reduced at wheat kernel structural conditions. The inactivation of <i>E. faecium</i> was better fitted in the Weibull distribution model for wheat dough structural conditions while in first-order kinetics for wheat kernel and flour structural conditions due to the changes of physical structures during heating. A better pasteurization effect could be achieved in wheat kernel structure in this study, which may provide technical support for thermal inactivation of pathogens in wheat-based food processing.