The observation of amyloid-β (Aβ) lesions using autofluorescence in transgenic mice and human Alzheimer disease patients has been reported frequently. However, no reports verify the autofluorescence of spontaneous Aβ amyloidosis in animals, to our knowledge. We validated the autofluorescence of Aβ lesions in spontaneous squirrel monkey cases under label-free conditions; lesions had intense blue-white autofluorescence in fluorescence microscopy using excitation light at 400–440 nm. Thioflavin S staining and immunohistochemistry of the same specimens revealed that this blue-white autofluorescence was derived from Aβ lesions. Hyperspectral analysis of these lesions revealed a characteristic spectrum with bimodal peaks at 440 and 460 nm, as reported for Aβ lesions in mice. Principal component analysis using hyperspectral data specifically separated the Aβ lesions from other autofluorescent substances, such as lipofuscin. A non-labeled and mechanistic detection of Aβ lesions by hyperspectral imaging could provide valuable insights for developing early diagnostic techniques.