Objective: To investigate the persistence of staining after the use of chlorhexidine gluconate mouthrinse. Method and Materials: Twenty-four subjects (nine women and 15 men) who underwent periodontal therapy and were prescribed the use of 0.2% chlorhexidine gluconate mouthrinse participated in this study. Color values of maxillary central incisors, canines, and first molars were recorded at baseline; 3 days; and 1, 2, and 3 weeks of twice-daily chlorhexidine gluconate use with a digital intraoral colorimeter according to the CIE L*a*b* coordinates. Results: While color-change (ΔE) values showed significant differences (P = .020) at different time points (10.1, 8.9, 8.9, 9.4, after 3 days and 1, 2, and 3 weeks, respectively), the duration of chlorhexidine gluconate use did not significantly affect the results (P = .873) (two-way ANOVA, Tukey test). No significant difference was found among ΔL* (P = .070), Δa* (P = .169), and Δb* (P = .691) values at any time point (one-way ANOVA). Measurements of baseline to day 3 differences showed significantly higher ΔE values than those at other time points (P .05), but this change remained nonsignificant after 1, 2, and 3 weeks of chlorhexidine gluconate use (P > .05) (Tukey test). The highest visible staining occurred on the first molars at all time points (83%, 79%, 79%, and 96% after 3 days and 1, 2, and 3 weeks, respectively) compared to the other teeth evaluated. Conclusion: The staining effect of chlorhexidine gluconate mouthrinse on natural dentition should be expected to be the highest in the first few days of use. Keywords: chlorhexidine, color, dental staining