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Distinguishing Crohn's disease (CD) from ulcerative colitis (UC) may be difficult when the disease is limited to the colon. Transmural healing is an important adjunctive measure of inflammatory bowel disease (IBD) activity. The aim of this study was to examine the role of endoscopic ultrasound (EUS) in differentiating CD versus UC and evaluating transmural disease activity.This prospective cohort enrolled 20 patients with CD (10 active, 10 inactive), 20 patients with UC (10 active, 10 inactive), and 20 controls who underwent colonoscopy from 2019 to 2021 at a tertiary care center. Measurements of bowel wall layer thickness from the rectum and cecum were obtained using a through-the-scope ultrasound catheter (Olympus UM-3R-3) at the time of colonoscopy.Compared to controls, patients with active CD (aCD) had thicker rectal submucosa and total wall layer (submucosa 1.80 [1.40-2.00] vs 0.60 [0.40-0.70] mm, total wall 3.70 [3.52-4.62] vs 2.10 [1.70-2.40] mm, respectively, P0.01). Similar significant findings were observed for the cecal wall layers. Compared to controls, patients with active UC (aUC) had thicker rectal mucosa and total wall but not submucosa nor muscularis propria layer (mucosa 1.35 [1.12-1.47] vs 0.60 [0.57-0.70] mm, total wall 3.45 [2.85-3.75] vs 2.10 [1.70-2.40] mm, respectively, P0.01). Patients with aCD compared to those with aUC had significantly thicker rectal submucosa layer (1.80 [1.40-2.00] vs 0.55 [0.40-0.75] mm, P0.01). Cut-off values of 1.1 mm for rectal submucosa in CD and 1.1 mm for rectal mucosa in UC can differentiate active from inactive disease (Sensitivity (Se) 1.0, Specificity (Sp) 1.0 and Se 0.8, Sp 0.9, respectively).EUS measurements of colon wall layers can help diagnose active CD versus UC and assess transmural disease activity.