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This study aimed to assess the effects of Penicillium expansum (P. expansum) infection on the quality and flavor of Jinmi (JM) and Jinyan (JY) kiwifruit. Kiwifruit were inoculated with P. expansum and stored at 0 ± 1°C, and the changes in quality indicators and volatile substances (VCs) at different stages of disease were determined. Results showed that in wound-inoculated kiwifruit, the soluble solid content (SSC) increased. Conversely, their titratable acidity and vitamin C (VC) content, firmness, lightness, and saturation decreased. The taste-related parameters and nutritional value of kiwifruit declined after infection. VCs such as ethanol, 3-methyl-1-butanol, and 2-methylisoborneol were detected only in the diseased fruit and gradually increased as the disease aggravated, suggesting that they may be the main sources of odor during P. expansum infection. Therefore, VCs detection can be used to determine possible P. expansum infection, as well as the degree of infection in kiwifruit. PRACTICAL APPLICATIONS: In practical application, we can use the results of this study to determine possible Penicillium expansum infection, as well as the degree of infection in kiwifruit according to the indicators such as volatile substances. Kiwifruit enterprises can use the nondestructive detection model established in this study to screen out the kiwifruit infected with P. expansum more efficiently, quickly, and accurately, in order to prevent harm to the health of consumers., (© 2021 Wiley Periodicals LLC.)