Purpose: Early diagnosis of tooth enamel demineralization, and dentin caries lesions, present a valuable solution to avoid or decrease their deleterious effect. The aim of this study was to design a simple, effective, and non-invasive technique, employing a novel laser wavelength to classify and differentiate between various tooth abnormalities in-vitro, by estimating wavelengths, showing distinctive appearance for each tooth class., Methods: This study implies a fluorescence hyperspectral imaging system employing a 395-nm laser diode source, irradiating a pre-diagnosed 12 molars and premolars teeth. The obtained reconstructed images were displayed and processed by HSAnalysis2XL, accompanied by a custom made digital, and image signal processing algorithms, revealing the exact wavelengths, characterizing the fluorescence of each tooth pre-diagnosed class., Results: The proposed hyperspectral imaging system was able to discriminate between normal, and abnormal dental classes for the entire specimens. Furthermore, a series of wavelengths, noting each lesion individually were obtained from the spectroscopic hyperspectral output. The root calculus, white spot, dentin caries, and enamel caries have a bright visual appearance at λ 3 = 702 nm, λ 5 = 771 nm, and λ 6 = 798 nm respectively. Consequently, these abnormalities exhibit a dark appearance at λ 1 = 421 nm, λ 2 = 462 nm, and λ 4 = 734 nm. The wavelength selections were confirmed by the grayscale image outcomes., Conclusions: This study provides a set of wavelengths that can be employed by dentists to diagnose white spot, root calculus, and enamel dentin caries lesions under the irradiation of a new UV-vis laser illumination source without, any hazardous thermal or mechanical effects., (Copyright © 2018 Elsevier B.V. All rights reserved.)