Tumor hypoxia is a major factor responsible for tumor progression, metastasis, invasion, and treatment resistance, leading to low local tumor control and recurrence after radiotherapy in cancers. Here,18F-positron emission tomography (PET) probes are developed for visualizing viable hypoxic cells in biopsies. Pimonidazole derivatives and nitroimidazole-based agents bearing sulfonyl linkers were evaluated. A small-animal PET study showed that the tumor uptake of [18F]-23 [poly(ethylene glycols) (PEG)-sulfonyl linker] of 3.36 ± 0.29%ID/g was significantly higher (P < 0.01) than that of [18F]-20 (piperazine-linker tracer, 2.55 ± 0.49%ID/g) at 2 h postinjection in UPPL tumors. The tumor-to-muscle uptake ratio of [18F]-23 (2.46 ± 0.48 at 2 h pi) was well improved compared with that of [18F]-FMISO (1.25 ± 0.14 at 2 h pi). A comparable distribution pattern was observed between ex vivo autoradiography of [18F]-23 and pimonidazole staining of the neighboring slice, indicating that [18F]-23 is a promising PET agent for hypoxia imaging.