The morphology of urothelial carcinomas, particularly when poorly differentiated or in metastatic sites, is not distinctive and overlaps significantly with other poorly differentiated nonurothelial carcinomas. Currently, there is no widely used single marker or panel of markers to confirm urothelial origin. We evaluated a panel consisting of antibodies to uroplakin III (UROIII), thrombomodulin (THR), high molecular weight cytokeratin (HMWCK), and cytokeratin 20 (CK20) in a wide range of urothelial tumors. Immunohistochemistry was performed on 112 paraffin-embedded urothelial neoplasms: 14 low malignant potential, 16 low-grade noninvasive, 16 high-grade noninvasive, 36 invasive, and 25 metastatic and 5 small cell carcinomas of the urinary bladder. Tissue microarray analysis was used to examine 498 tissue cores of nonurothelial tumors and normal tissue using antibodies to UROIII, THR, and HMWCK. Overall positive staining results in all urothelial tumors are as follows: UROIII, 64 of 112 (57.1%); THR, 77 of 112 (68.8%); HMWCK, 88 of 110 (80%); and CK20, 53 of 110 (48.2%). The expression of the four markers varied with tumor grade and stage. All small cell carcinomas were negative for all markers. Variant morphologic subtypes showed similar staining as conventional urothelial carcinomas. Tissue microarray analysis showed no UROIII immunoreactivity in tissue cores of nonurothelial tumors. THR was expressed by a limited number of nonurothelial cores (10 of 37 [27%] non-small cell lung carcinomas, 2 of 36 [5.6%] lymphomas). HMWCK was expressed by 43.8% of non-small cell lung carcinomas and essentially absent in other nonurothelial tumor cores. Based on the results of the study, the expression of UROIII in a tumor is essentially diagnostic of urothelial origin; however, it is expressed in only slightly more than half of urothelial tumors. The coexpression of THR, HMWCK, and CK20 strongly suggests urothelial origin. The coexpression of two of three non-UROIII markers (THR, HMWCK, CK20) suggests urothelial origin but requires clinicopathologic correlation. The results of the study indicate a role for an antibody panel that includes UROIII, THR, HMWCK, and CK20 in the diagnosis of urothelial tumors.