Increasing evidence suggests that oxidative stress is associated with normal aging and several neurodegenerative diseases, including Alzheimer's disease (AD). Here we quantified multiple oxidized bases in nuclear and mitochondrial DNA of frontal, parietal, and temporal lobes and cerebellum from short postmortem interval AD brain and age‐matched control subjects using gas chromatography/mass spectrometry with selective ion monitoring (GC/MS‐SIM) and stable labeled internal standards. Nuclear and mitochondrial DNA were extracted from eight AD and eight age‐matched control subjects. We found that levels of multiple oxidized bases in AD brain specimens were significantly (p < 0.05) higher in frontal, parietal, and temporal lobes compared to control subjects and that mitochondrial DNA had approximately 10‐fold higher levels of oxidized bases than nuclear DNA. These data are consistent with higher levels of oxidative stress in mitochondria. Eight‐hydroxyguanine, a widely studied biomarker of DNA damage, was approximately 10‐fold higher than other oxidized base adducts in both AD and control subjects. DNA from temporal lobe showed the most oxidative damage, whereas cerebellum was only slightly affected in AD brains. These results suggest that oxidative damage to mitochondrial DNA may contribute to the neurodegeneration of AD.