We examined diagnostic variation of flat intraurothelial lesions with comparison with immunohistochemical and fluorescence in situ hybridization (FISH) analyses. Nine uropathologists diagnosed 23 biopsy samples from the urinary bladder. The samples were analyzed by immunohistochemical expression of cytokeratin 20, high-molecular-weight cytokeratin, Ki-67, p53, and p16(INK4a), and multicolor FISH using the UroVysion probe set (Vysis, Abbott, Des Plaines, IL). Diagnostic agreement for each classification and for nonneoplastic or neoplastic lesions was obtained in 8 (35%) and 16 (70%) of 23 lesions, respectively. The preference ratio of neoplasia to nonneoplasia (0.9 to 4.8) or carcinoma in situ to dysplasia (0.2 to 4.0) also varied among the pathologists. In 6 ancillary analyses, the majority of neoplastic lesions with diagnostic agreement indicated more than 2 aberrant results, whereas the majority of lesions without diagnostic agreement showed no or only 1 aberrant result. The molecular and immunohistochemical analyses can discriminate between neoplastic and nonneoplastic lesions; however, they cannot reliably solve diagnostic variation of flat intraepithelial lesions.