Purpose: To evaluate the histological characteristics of buccal mucosa samples prepared using different dissection techniques. Methods: The buccal mucosae harvested from 9 men submitted to a substitution urethroplasty were used in this study. Biopsy specimens of buccal mucosae were grouped according to the macroscopic appearance after dissection: Group 1 – yellowish appearance (minimal dissection), Group 2 – whitish appearance (more aggressive dissection), and Group 3 – translucent appearance (ultra-aggressive dissection). The biopsy specimens were evaluated using histomorphometric techniques. The thicknesses of the graft, epithelium, and sub-epithelial connective tissue were compared. Results: The samples from Group 3 and Groups 2 and 3 presented lower total and lower sub-epithelial connective tissue thickness, respectively, when compared with Group 1. The epithelial thickness was not affected by the dissection method. Muscular and adipose tissues were present in 77 and 55% of samples from Group 1, respectively, whereas these were observed in 44 and 22% of samples from Group 2 respectively. The muscular tissue was observed in 11% of the cases, and the adipose tissue was not observed in samples prepared with ultra-aggressive dissection. Conclusion: We conclude that an intermediate graft dissection (whitish appearance, as performed in Group 2) presents the best balance between subepithelial connective tissue preservation and adipose and muscle tissue removal.