Urothelial carcinoma in situ (CIS) is a high-grade neoplasm and an indicator of recurrence and progression that requires specific treatment. The distinction of CIS from flat non-neoplastic urothelium, in particular dysplasia, on the basis of histologic features is often difficult, and this study aims to validate cytokeratin 20 (CK20) and Ki-67 as discriminatory markers for this purpose. Immunostaining of these markers was applied to 26 cases of CIS, 14 atypia of unknown significance, 4 dysplasia, 6 normal, and 9 hyperplastic urothelium. CIS showed CK20 staining of deep urothelial cells in 23/26 CIS compared with restricted staining in surface cells in all non-neoplastic lesions. CIS had significantly increased Ki-67 index with a mean of 53.37% compared with that of non-neoplastic urothelium, which was <10% (P<0.0001). The proliferating cells were distributed randomly in CIS, whereas in non-neoplastic urothelium, staining was confined to the basal layer. Among the cases of atypia, 3/14 displayed deep staining for CK20 and 6/14 had elevated Ki-67 counts. In dysplasia similar findings were present in 1/4 and 2/4 cases, respectively. These findings suggest that CK20 and Ki-67 are objective markers to distinguish CIS from non-neoplastic urothelium. In cases of "atypia of unknown significance" and "dysplasia," positivity for both markers should raise the possibility of CIS or preneoplastic change and identify those cases for follow-up.