Abstract Objectives We studied the diagnostic accuracy and discordance of upper tract urothelial carcinoma (UTUC) by comparing biopsy and urinary cytology with matched nephroureterectomy specimens. Methods Sixty-nine patients with UTUC without neoadjuvant treatment were retrospectively identified who had matched biopsy and nephroureterectomy specimens. Twenty patients had concurrent upper tract cytology. H&E and cytology slides were re-reviewed. Statistical analysis was performed. Results Patients included 48 men and 21 women with a mean age of 69 years. A concordant grade between biopsy and surgical specimen was present in 49 (71%) patients. The mean size of biopsy specimens in the discordant group was significantly smaller than that in the concordant group. Invasion was evaluated in 48 biopsy cases that had adequate subepithelial tissue, and 33 of them were diagnosed with concordant invasion status. Mean tumor size in both tumor grade and invasion discordant groups was significantly larger than that in the concordant group. High-grade urothelial carcinoma was detected in 84% of cases using urinary cytology. Conclusions Our study demonstrates the diagnostic challenges of UTUC on small biopsy specimens. Biopsy specimen size and tumor size are significantly associated with the diagnostic discordance. Upper tract cytology showed high diagnostic accuracy and should be complementary to the biopsy.