Since cholinergic dysfunction has been implicated in Alzheimer's disease (AD), the effects of Aβ plaques on nicotinic acetylcholine receptors (nAChRs) α4β2* subtype were studied using the transgenic 5xFAD mouse model of AD. Using the PET radiotracer [18F]nifene for α4β2* nAChRs, in vitro autoradiography and in vivo PET/CT studies in 5xFAD mice were carried out and compared with wild‐type (C57BL/6) mice. Ratios of [18F]nifene binding in brain regions versus cerebellum (CB) in 5xFAD mice brains were for thalamus (TH) = 17, hippocampus‐subiculum = 7, frontal cortex (FC) = 5.5, and striatum = 4.7. [125I]IBETA and immunohistochemistry (IHC) in 5xFAD brain slices confirmed Aβ plaques. Nicotine and acetylcholine displaced [18F]nifene in 5xFAD mice (IC50 nicotine = 31–73 nM; ACh = 38–83 nM) and C57BL/6 (IC50 nicotine = 16–18 nM; ACh = 34–55 nM). Average [18F]nifene SUVR (CB as reference) in 5xFAD mice was significantly higher in FC = 3.04 compared to C57BL/6 mice FC = 1.92 (p = .001), whereas TH difference between 5xFAD mice (SUVR = 2.58) and C57BL/6 mice (SUVR = 2.38) was not significant. Nicotine‐induced dissociation half life (t1/2) of [18F]nifene for TH were 37 min for 5xFAD mice and 26 min for C57BL/6 mice. Dissociation half life  for FC in C57BL/6 mice was 77 min , while no dissociation of [18F]nifene occurred in the medial prefrontal cortex (mFC) of 5xFAD mice. Coregistration of [18F]nifene PET with MR suggested that the mPFC, and anterior cingulate (AC) regions exhibited high uptake in 5xFAD mice compared to C57BL/6 mice. Ex vivo [18F]nifene and in vitro [125I]IBETA Aβ plaque autoradiography after in vivo PET/CT scan of 5xFAD mouse brain were moderately correlated (r2 = 0.68). In conclusion, 5xFAD mice showed increased non‐displaceable [18F]nifene binding in mPFC.