Purpose/Hypothesis: In patients with Painful Bladder Syndrome (PBS) to determine whether palpable pelvic floor muscular abnormalities are identifiable on 3-dimensional (3-D) MRI imaging. Number of Subjects: Five subjects participated in this pilot study: three with PBS and two pain-free controls. Materials/Methods: Three women with PBS and two asymptomatic controls underwent a standardized musculoskeletal pelvic girdle assessment by the study physical therapist and underwent MRI according to a standard protocol and 3-D MRI images were constructed from the 2-D images. Results: On physical examination, control subjects were tender to palpation a mean of 1.0 out of 12 hip girdle muscles and PBS subjects were tender in a median of 7 of 12 muscles. Tenderness to palpation of the pelvic floor muscles scored a mean of 21.6 out of a possible 30 in the PBS group and 1.0 out of 30 in the control group. Trigger points and tight palpable bands were present in an average of 1.5 sites in the control group and 8.7 sites in the PBS out of a possible 10 sites. All participants had negative Trandelenburg, posterior pelvic pain provocation and forced FABERs tests. The MRI images revealed striking morphologic differences between PBS patients and controls: PBS patients had larger muscle mass and V-shaped cross-section. Controls had U-shaped levator muscle bodies. Conclusions: This pilot study suggests that the palpable pelvic floor muscular abnormalities can be visualized using MRI. Clinical Relevance: We hope that these findings will prompt clinicians to strongly consider that the pain of PBS might arise within the pelvic floor musculature, and will prompt referral to physical therapy for appropriate treatment. It also gives insight into the nature of those muscular abnormalities, perhaps enabling us to better define and communicate our findings with one another.