Objective We investigated the potential of [¹⁸F]fluorodeoxyglucose ([¹⁸F]FDG) and [¹⁸F]Fluoromethylcholine ([¹⁸F]FCho) PET, compared to contrast-enhanced MRI, for the early detection of treatment response in F98 glioblastoma (GB) rats. Methods When GB was confirmed on T2- and contrast-enhanced T1-weighted MRI, animals were randomized into a treatment group (n = 5) receiving MRI-guided 3D conformal arc micro-irradiation (20 Gy) with concomitant temozolomide, and a sham group (n = 5). Effect of treatment was evaluated by MRI and [¹⁸F]FDG PET on day 2, 5, 9 and 12 post-treatment and [¹⁸F]FCho PET on day 1, 6, 8 and 13 post-treatment. The metabolic tumor volume (MTV) was calculated using a semi-automatic thresholding method and the average tracer uptake within the MTV was converted to a standard uptake value (SUV). Results To detect treatment response, we found that for [¹⁸F]FDG PET (SUV_mean x MTV) is superior to MTV only. Using (SUV_mean x MTV), [¹⁸F]FDG PET detects treatment effect starting as soon as day 5 post-therapy, comparable to contrast-enhanced MRI. Importantly, [¹⁸F]FDG PET at delayed time intervals (240 min p.i.) was able to detect the treatment effect earlier, starting at day 2 post-irradiation. No significant differences were found at any time point for both the MTV and (SUV_mean x MTV) of [¹⁸F]FCho PET. Conclusions Both MRI and particularly delayed [¹⁸F]FDG PET were able to detect early treatment responses in GB rats, whereas, in this study this was not possible using [¹⁸F]FCho PET. Further comparative studies should corroborate these results and should also include (different) amino acid PET tracers.