Atypical urine cytology (CYT) triggers a cystoscopic or another ancillary investigation that targets urothelial neoplasms. We report a case presenting as an unknown primary malignancy, which illustrated the diagnostic utility of direct double immunostaining for cytokeratin 20 (CK20)/p53 expression in a urine CYT specimen. A 42-year-old woman visited the emergency room for pain in her right lower abdominal quadrant. Computed tomography revealed postrenal obstructive hydronephrosis, and her urine CYT showed malignancy, type undetermined. Atypical cells that are positive for cytoplasmic expression of CK20 and nuclear expression of p53 could facilitate the decision to perform a nephroureterectomy for urothelial carcinoma.