To noninvasively observe dynamic changes in tumor hypoxia in mouse models of human non-small-cell lung cancer (NSCLC) using ¹⁸F-fluoromisonidazole PET. Nude mice with NSCLC H460 and A549 subcutaneous xenografts were coinjected intravenously with ¹⁸F-fluoromisonidazole and the hypoxia marker pimonidazole, and observed by serial PET scans. After sacrifice, the tumor distribution of ¹⁸F-fluoromisonidazole and pimonidazole was compared by digital autoradiography and microscopy, respectively. The NSCLC hypoxic microenvironment was spatially heterogeneous. Serial PET scans over 48 h revealed an apparent change in the intratumoral distribution of ¹⁸F-fluoromisonidazole. The tumor hypoxic microenvironment is spatially and temporally heterogeneous, and hypoxic cancer cells have a shorter life span when growing in vivo. Therefore, the concept of hypoxic resistance and hypoxia-targeting therapy of macroscopic tumors should be revisited.