Objective: To study the clinicopathologic features of urothelial hyperplastic lesion with an endophytic growth pattern and the role of immunohistochemistry and multitargeted fluorescence in situ hybridization (FISH) in the differential diagnosis., Methods: Forty-one cases of urothelial lesions exhibiting endophytic growth patterns were reviewed and reclassified as inverted papilloma, urothelial carcinoma with an endophytic growth pattern, and florid von Brunn nest. The gains of chromosomes 3, 7, and 17 and loss of 9p21 was detected by FISH, and performed immunohistochemical staining for CK20, p53, and Ki-67. Follow-up data of 12 cases were obtained., Results: (1) Twelve inverted papillomas sized 1.2 cm in average, consisted of anastomosing cords and nests with uniform width distribution involving the lamina propria, the central portion contained streaming cells with squamous metaplasia, and the periphery showed palisading. No or rare atypia and mitosis were found. Focal exophytic papillary component lined by less than 6 layers of normal urothelium were observed in 4 cases. (2) Twenty-four urothelial carcinomas with an endophytic growth pattern sized 2.1 cm in average, demonstrated the similar architecture with inverted papilloma, but exhibited thick columns and variable thickness of the cords, irregular size and shape of large nests with transition into solids. Mild to moderate cytologic atypia was shown, and mitotic figures ranged 1 to 8 per 10 HPFs. Exophytic papillary component was not observed in 3 cases, but the superficial urothelium showed dysplasia, while coexisted exophytic component in other cases was associated with low malignant potential or low grade tumor. (3) Five florid von Brunn nests sized 0.9 cm in average, had normal or hyperplastic urothelium, variable nests with cysts compacted in lamina propria, no cytologic atypia and mitosis. Twenty-one of 24 (79.1%) urothelial carcinomas with an endophytic growth pattern displayed abnormally positive results by multitargeted FISH, whereas all inverted papillomas and florid von Brunn nests were negative. Immunohistochemically, CK20 was weakly positive in 2 cases of urothelial carcinoma with an endophytic growth pattern, and negative in all inverted papillomas and florid von Brunn nests. p53 weakly stained 5% to 50% nuclei of the tumor cells in 16 cases of urothelial carcinomas with an endophytic growth pattern and 1 inverted papilloma. 1%-5% tumor cells expressed Ki-67 in urothelial carcinoma with an endophytic growth pattern, and less than 1% in inverted papilloma and florid von Brunn nests. Follow-up study revealed that 2 cases of urothelial carcinoma with an endophytic growth pattern had developed invasive carcinoma, underwent cystectomy, and metastasized remotely. No recurrence occurred in cases of inverted papilloma., Conclusions: Benign and malignant urothelial lesions with an endophytic growth pattern present histologic overlapping. Urothelial carcinoma with an endophytic growth pattern displays unique characteristics in morphology and immunohistochemistry. Multitargeted FISH analysis is helpful in the differential diagnosis.