In response to double-stranded breaks (DSBs) in chromosomal DNA, H2AX (a member of histone H2A family) becomes phosphorylated to form γH2AX. Although increased levels of γH2AX have been reported in the neuronal nuclei of Alzheimer’s disease (AD) patients, the understanding of γH2AX responses in buccal nuclei of individuals with mild cognitive impairment (MCI) and AD remain unexplored. In the current study, endogenous γH2AX was measured in buccal cell nuclei from MCI (n = 18) or AD (n = 16) patients and in healthy controls (n = 17) using laser scanning cytometry (LSC). The γH2AX level was significantly elevated in nuclei of the AD group compared to the MCI and control group, and there was a concomitant increase in P-trend for γH2AX from the control group through MCI to the AD group. Receiver-operating characteristic curves were carried out for different γH2AX parameters; γH2AX in nuclei resulted in the greatest area under the curve value of 0.7794 (p = 0.0062) with 75% sensitivity and 70% specificity for the identification of AD patients from control. In addition, nuclear circularity (a measure of irregular nuclear shape) was significantly higher in the buccal cell nuclei from the AD group compared with the MCI and control groups. Additionally, there was a positive correlation between the nuclear circularity and γH2AX signals. The results indicated that increased DNA damage is associated with AD.