Statement of problem: The color of vital craniofacial structures has not been measured accurately. Purpose: The purpose of this study was to determine the color of vital craniofacial structures and evaluate the validity and test-retest reliability of a noncontacting 45/0-degree optical configuration. Material and methods: A spectroradiometer and an external light source were configured in a noncontacting 45/0-degree (45-degree illumination and 0-degree observer) optical configuration to measure the color of subjects' vital craniofacial structures (central and lateral incisor and canine, attached gingiva, lips, and facial skin). The 120 subjects were stratified into 5 age groups with 4 racial categories and balanced for gender. For evaluation of validity, linear regressions and 95% confidence intervals were calculated for ΔL*, Δa*, Δb* [color difference of (CIE) LAB values] between the measured and certified values of the 22 color patches of the DC Color Checker. For test-retest reliability, a random sample of 12 (10%) subjects was remeasured at a second visit. Paired t tests, correlations, and Bland-Altman analyses were performed between the first and second measurements of the 12 pairs of L*, a*, and b* values for the 6 craniofacial structures. Results: For validity, the mean color difference and linear regression for Commission Internationale d'Eclairage (CIE) LAB values between measured and certified color of the 22 opaque color patches were ΔE of 1.46 and 0.99 for all regressions, respectively. Only Δa* did not contain zero in its 95% confidence interval. For test-retest reliability, no paired t tests were significantly different from each other, and the Pearson correlation coefficient ranged from 0.9 (9 pairs) to 0.7 (3 pairs). Ten of the 18 Bland-Altman plots showed good reliability. Conclusion: The spectral reflectance of craniofacial structures can be measured with acceptable validity and test-retest reliability using a noncontacting 45/0-degree optical configuration.