OBJECTIVES: To compare the histological features, biomechanical properties, water permeability, and osmotic stress responses of human buccal mucosa and penile skin flaps to evaluate their suitability as urethral graft substitutes. METHODS: Buccal mucosa (n = 53) and penile skin flap (n = 59) were prepared for histological assessment of tissue structural characteristics and vascular patterns using H&E staining and immunofluorescence. Biomechanical properties were evaluated through mechanical testing, water permeability through deuterium oxide diffusion assays, and osmotic stress responses under hypo-, iso-, and hypertonic conditions. RESULTS: H&E staining revealed that the buccal mucosa epithelium was significantly thicker (628.8 ± 213.3 μm) than that of the penile skin flap (148.2 ± 62.1 μm
p <
0.05). Mechanical testing demonstrated that the buccal mucosa had significantly higher elastic modulus and tensile strength and lower tensile strain compared with penile skin flap (p <
0.05). Water permeability testing revealed that the cumulative diffusion of deuterium oxide (D CONCLUSION: This study presents the first comprehensive comparison of the tissue properties of buccal mucosa and penile skin flaps. Our findings provide valuable insights for surgical decision-making, postoperative management, personalized treatment strategies, and the advancement of bioengineered urethral substitutes.