Accurate recognition of muscularis propria invasion by urothelial carcinoma is vital as it serves as a crossroad between conservative and aggressive clinical management. Recently, there has been attention to the hyperplastic pattern of muscularis mucosae which may mimic the muscularis propria. We have earlier shown that smoothelin, a marker of terminally differentiated smooth muscle cells, is relatively specific for muscularis propria (positive staining) and is variably negative to weak in muscularis mucosae. The earlier study was based on cystectomy specimen slides in which the bladder cancer was not present. Pathologic staging in transurethral resection of urinary bladder tumor (TURBT) specimens is complicated by limited, unoriented, or highly cauterized samples. Herein, we test the capability of smoothelin to recognize muscularis propria in TURBT specimens to further substantiate its diagnostic applicability in routine practice. Representative sections from 70 TURBTs were immunostained with smoothelin, and muscularis propria was evaluated in H&E slides and the corresponding smoothelin immunohistochemistry slides using double-blinded analysis. In 31/70 (44%) cases, muscularis propria was involved by invasive carcinoma. Cautery artifact was present in 46/70 (66%) cases, which did not seem to affect smoothelin immunohistochemistry staining of the muscularis propria. Muscularis propria was present by H&E in 48/70 (69%) cases and 48/70 (69%) cases had muscularis propria by smoothelin immunohistochemistry-based 2 (+) or 3 (+) positivity in larger muscle bundles with round regular contours. Desmoplastic response to invasive carcinoma stained negatively for smoothelin. The sensitivity, specificity, positive predictive value, and negative predictive value of smoothelin based on comparison with morphology in TURBT specimens was 98%, 95%, 98%, and 95%, respectively. This study confirms the relatively high sensitivity and specificity for smoothelin in MP, including in TURBT specimens. Immunoreactivity is retained despite the presence of thermal tissue injury, desmoplasia, or involvement by carcinoma. Our data confirm the use of smoothelin in the accurate distinction between muscularis propria and muscularis mucosae or desmoplastic reactions, thereby facilitating appropriate pathologic stage designation in often challenging TURBT specimens.