Purpose To elucidate the molecular pathogenesis of age-related macular degeneration (AMD) and interpretation of fundus autofluorescence (AF) imaging, we identified spectral AF characteristics of drusen and retinal pigment epithelium (RPE) in donor eyes with AMD. Methods Macular RPE/Bruch’s membrane (BrM) flat mounts were prepared from 5 donor eyes with AMD. In 12 locations (1–3/eye), hyperspectral AF images in 10-nm-wavelength steps were acquired at 2 excitation wavelengths (λex 436 nm, 480 nm). A non-negative tensor factorization algorithm was used to recover 5 abundant emission spectra and their corresponding spatial localizations. Results At λex 436 nm, we consistently localized a novel spectrum (SDr) with a peak emission near 510 nm in drusen and sub-RPE deposits. Abundant emission spectra seen previously (S0 in BrM and S1, S2, and S3 in RPE lipofuscin/melanolipofuscin (L/ML), respectively), also appeared in AMD eyes, with the same shapes and peak wavelengths as in normal tissue. L/ML spectra localizations in AMD eyes varied widely in their overlap with drusen, ranging from none to complete. Conclusions An emission spectrum peaking at ~510 nm (λex 436 nm) appears to be sensitive and specific for drusen and sub-RPE deposits. One or more abundant spectra from RPE organelles exhibit characteristic relationships with drusen.