Context .- Urothelial carcinoma in situ (CIS) is a precursor of invasive bladder cancer, which if left untreated, will likely progress to more aggressive disease. Approximately 50% of CIS lesions are missed on routine cystoscopy owing to their flat architecture. Furthermore, many benign but abnormal-appearing areas may be biopsied owing to lack of cellular resolution of cystoscopes. Multiphoton microscopy (MPM) is an optical imaging technique that generates subcellular-resolution three-dimensional images from unfixed tissue without using exogenous dyes. Objective .- To assess the diagnostic potential of MPM in identifying and differentiating benign from malignant flat bladder lesions, especially CIS. Design .- Seventy-eight specimens (benign = 46, CIS = 23, invasive = 9, as diagnosed on histopathology) were obtained from flat bladder mucosa via transurethral resection of bladder, cold cup biopsy, or cystectomy, imaged fresh with a commercial benchtop MPM, and submitted for routine histopathology. Multiphoton microscopy and hematoxylin-eosin diagnoses were compared. Results .- In 77 of 78 specimens (99%), accurate MPM diagnoses (benign/malignant) were given on the basis of their architectural and cytologic features (nuclear to cytoplasmic ratio, pleomorphism, polarity/organization of urothelial layers, etc). The sensitivity and specificity were 97% and 100%, respectively, with positive (malignant) and negative (benign) predictive values of 100% and 98%, respectively. The interobserver agreement, κ, was 0.93. Conclusions .- Our study demonstrates the capability of MPM to identify and differentiate benign from malignant flat bladder lesions, especially CIS. With the advent of MPM endoscopes, we foresee their potential as a biopsy guidance tool for early detection and treatment of CIS, thus reducing the rate of biopsies with benign diagnoses and their associated complications.