The objective of this study is to examine observer agreement on the rank of the color tones after toluidine blue staining of a mucosal lesion. Cohort study with repeated measures is the design of the study. Twenty observers ranked and scored 8 specified areas on the color images of a lesion before and after toluidine blue application in two sessions. Inter and intra-observer variations were analyzed with Cohen's kappa. The L* (the black-white axis), a* (red-green axis), and b* (yellow-blue axis) values were measured and set as the gold standards. Intra and inter-observer agreements were D(0)aEuro parts per thousand= 0.86 and D-0 = 0.854. All color parameters were effective on the color ranking order (pL* = 0.00, pa* = 0.007, pb* = 0.00), although L* and b* were more effective on the ranking of the samples than a*. Areas that appeared pale blue visually had a significant blue component, but the observers were confused with the effect of whiteness of the area in clinical decision making.