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Abstract Hyperspectral data encode information from electromagnetic radiation (i.e., color) of any object in the form of a spectral signature; these data can then be used to distinguish among materials or even map whole landscapes. Although hyperspectral data have been mostly used to study landscape ecology, floral diversity and many other applications in the natural sciences, we propose that spectral signatures can be used for rapid assessment of faunal biodiversity, akin to DNA barcoding and metabarcoding. We demonstrate that spectral signatures of individual, live fish specimens can accurately capture species and clade-level differences in fish coloration, specifically among piranhas and pacus (Family Serrasalmidae), fishes with a long history of taxonomic confusion. We analyzed 47 serrasalmid species and could distinguish spectra among different species and clades, with the method sensitive enough to document changes in fish coloration over ontogeny. Herbivorous pacu spectra were more like one another than they were to piranhas; however, our method also documented interspecific variation in pacus that corresponds to cryptic lineages. While spectra do not serve as an alternative to the collection of curated specimens, hyperspectral data of fishes in the field should help clarify which specimens might be unique or undescribed, complementing existing molecular and morphological techniques.