['We performed a head-to-head comparison of an antibody against uroplakin III (UP3) and a new uroplakin II (UP2) antibody that remains untested in diagnostically challenging settings.', 'We immunostained high-grade bladder neck carcinomas (n = 35), high-grade upper tract urothelial carcinomas (UC) and renal carcinomas (n = 85), metastases of UC (n = 30) and a multicancer tissue microarray (n = 88) for UP3 and UP2, and scored staining intensity and proportion. UP3 showed membranous plaque-like expression, while UP2 staining showed both membranous and cytoplasmic positivity. Significantly greater intensity (P = 0.003) and proportion (P = 0.03) of staining was noted for UP2 among bladder neck lesions, with UP2 staining showing greater sensitivity (63% versus 19%) and similar specificity (95% versus 100%) for UC over prostate carcinoma (P = 0.02). Among upper tract lesions, UP2 staining showed greater intensity and proportion than UP3 (both P < 0.001), including improved sensitivity (68% versus 23%) and equal specificity (both 100%) for UC (P = 0.006). Among UC metastases, UP2 staining showed greater intensity and proportion (both P < 0.001) with higher sensitivity (73% versus 37%, respectively, P = 0.001). Of 88 additional cases tested, no non-urothelial cases stained for either UP.'] Does uroplakin II outperform uroplakin III in diagnostically challenging settings?