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A full comprehension of colorimetric relationships within and between teeth is key for aesthetic success of a dental restoration. In this sense, hyperspectral imaging can provide point-wise reliable measurements of the tooth surface, which can serve for this purpose. The aim of this study was to use a hyperspectral imaging system for the colorimetric characterization of 4 in-vivo maxillary anterior teeth and to cross-check the results with similar studies carried out with other measuring systems in order to validate the proposed capturing protocol. Hyperspectral reflectance images (Specim IQ), of the upper central (UCI) and lateral incisors (ULI), were captured on 30 participants. CIE-L*a*b* values were calculated for the incisal (I), middle (M) and cervical (C) third of each target tooth. <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><mo>Δ</mo><msubsup><mi>E</mi><mrow><mi>a</mi><mi>b</mi></mrow><mo>*</mo></msubsup></mrow></semantics></math></inline-formula> and <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><mo>Δ</mo><msub><mi>E</mi><mn>00</mn></msub></mrow></semantics></math></inline-formula> total color differences were computed between different tooth areas and adjacent teeth, and evaluated according to the perceptibility (PT) and acceptability (AT) thresholds for dentistry. Non-perceptible color differences were found between UCIs and ULIs. Mean color differences between UCI and ULI exceeded AT (<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><mo>Δ</mo><msubsup><mi>E</mi><mrow><mi>a</mi><mi>b</mi></mrow><mo>*</mo></msubsup></mrow></semantics></math></inline-formula> = 7.39–7.42; <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><mo>Δ</mo><msub><mi>E</mi><mn>00</mn></msub></mrow></semantics></math></inline-formula> = 5.71–5.74) in all cases. Large chromatic variations between I, M and C areas of the same tooth were registered (<inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><mo>Δ</mo><msubsup><mi>E</mi><mrow><mi>a</mi><mi>b</mi></mrow><mo>*</mo></msubsup></mrow></semantics></math></inline-formula> = 5.01–6.07 and <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><mo>Δ</mo><msub><mi>E</mi><mn>00</mn></msub></mrow></semantics></math></inline-formula> = 4.07–5.03; <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><mo>Δ</mo><msubsup><mi>E</mi><mrow><mi>a</mi><mi>b</mi></mrow><mo>*</mo></msubsup></mrow></semantics></math></inline-formula> = 5.80–8.16 and <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><mo>Δ</mo><msub><mi>E</mi><mn>00</mn></msub></mrow></semantics></math></inline-formula> = 4.37–5.15; and <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><mo>Δ</mo><msubsup><mi>E</mi><mrow><mi>a</mi><mi>b</mi></mrow><mo>*</mo></msubsup></mrow></semantics></math></inline-formula> = 5.42–5.92 and <inline-formula><math xmlns="http://www.w3.org/1998/Math/MathML" display="inline"><semantics><mrow><mo>Δ</mo><msub><mi>E</mi><mn>00</mn></msub></mrow></semantics></math></inline-formula> = 3.87–4.16 between C and M, C and I and M and I, respectively). The use of a hyperspectral camera has proven to be a reliable and effective method for color evaluation of in-vivo natural teeth.