Objective We sought to evaluate candidate mechanisms underlying the pelvic floor dysfunction in women with chronic pelvic pain (CPP) and/or painful bladder syndrome (PBS)/interstitial cystitis. Notably, prior studies have not consistently controlled for potential confounding by psychological or anatomical factors. Study Design As part of a larger study on pelvic floor pain dysfunction and bladder pain sensitivity, we compared a measure of mechanical pain sensitivity, pressure pain thresholds (PPTs), between women with pelvic pain and pain-free controls. We also assessed a novel pain measure using degree and duration of postexam pain aftersensation, and conducted structural and functional assessments of the pelvic floor to account for any potential confounding. Phenotypic specificity of pelvic floor measures was assessed with receiver operator characteristic curves adjusted for prevalence. Results A total of 23 women with CPP, 23 women with PBS, and 42 pain-free controls completed the study. Women with CPP or PBS exhibited enhanced pain sensitivity with lower PPTs (1.18 interquartile range, 0.87–1.41 kg/cm2 ) than pain-free participants (1.48 1.11-1.76 kg/cm2 ; P < .001) and prolonged pain aftersensation (3.5 0-9 vs 0 0-1 minutes; P < .001). Although genital hiatus ( P < .01) was wider in women with CPP there were no consistently observed group differences in pelvic floor anatomy, muscle tone, or strength. The combination of PPTs and aftersensation duration correlated with severity of pelvic floor tenderness (R2 , 41-51; P < .01). Even after adjustment for prevalence, the combined metrics discriminated pain-free controls from women with CPP or PBS (area under the curve, 0.87). Conclusion Both experimental assessment of pelvic floor pain thresholds and measurement of sustained pain are independently associated with pelvic pain phenotypes. These findings suggest systematic clinical assessment of the time course of provoked pain symptoms, which occurs over seconds for mechanical pain thresholds vs minutes for aftersensation pain, would be helpful in identifying the fundamental mechanisms of pelvic floor pain. Longitudinal studies of therapies differentially targeting these discrete mechanisms are needed to confirm their clinical significance.