Objective To estimate the accuracy of bladder wall thickness (BWT), measurement by transvaginal bladder ultrasound (BUS) in diagnosing detrusor overactivity (DO) Methods A multicenter study in 687 women with overactive bladder or urgency predominant mixed incontinence in 22 centres was undertaken. Index test was BWT and urodynamics was the reference standard. Reproducibility of BWT and the acceptability of BWT and urodynamics were also studied. Results BWT showed poor sensitivity, specificity and likelihood ratios at all pre-specified cut-offs. The ROC curve showed no evidence of discrimination at any threshold between those with and without DO (p=0.25): the AUC was 0.53, 95%CI: (0.48, 0.57). In the prospective interobserver variation study of scans performed by two independent observers, the SD 0.95mm with an analytical variability of 0.76mm. There was a trend towards greater acceptability of BWT scan compared to urodynamics (p<0.001), (81% versus 56%). Conclusion Bladder wall thickness measurement did not discriminate women with DO versus those without DO and hence is not an accurate test for diagnosing DO. Transvaginal BWT does not have sufficient reliability and reproducibility. Women experienced higher levels of embarrassment and a lower rate of acceptability with urodynamics compared to the BWT scan procedure.