OBJECTIVE: This study was undertaken to develop and test a 3-dimensional (3D) color thickness mapping technique on levator ani imaged with magnetic resonance imaging (MRI). METHODS: Supine MRI datasets from 30 women were studied: 10 asymptomatic, 10 with urodynamic stress incontinence, and 10 with pelvic organ prolapse. Levators were manually outlined, and thickness mapping applied. Three-dimensional models were colored topographically, reflecting levator thickness. Thickness and occurrences of absent levator substance (gaps) were compared across the 3 groups, using nonparametric statistical tests. RESULTS: Color thickness mapping was successful in all subjects. There were statistically significant differences in thickness and gap percentages among the 3 groups of women, with thicker, bulkier levators in asymptomatic women, compared with women with prolapse or urodynamic stress incontinence. CONCLUSION: Color thickness mapping is feasible. It may be used to compare levators in symptomatic and asymptomatic women, to study relationships between levator thickness and pelvic floor dysfunction. This technique can be used in larger studies for hypothesis testing.