Dentinal surfaces prepared with air abrasion have considerably different characteristics from those prepared with conventional instruments. Different hybrid layer morphology and thickness occur, which can result in differences in the quality of restorations placed on dentinal surfaces prepared with a diamond bur compared to surfaces prepared using air abrasion. The objective of this study was to compare the hybrid layer thickness and morphology formed utilizing Scotchbond Multi-Purpose Plus (SBMP) on dentin prepared with a diamond bur in a high-speed handpiece and on dentin prepared using air abrasion. Flat dentin surfaces obtained from five human teeth were prepared using each method, then treated with the dentin adhesive system according to manufacturer's instructions. After a layer of composite was applied, specimens were sectioned, flattened, polished, and prepared for scanning electron microscopy. Ten different measurements of hybrid layer thickness were obtained along the bonded surface in each specimen. SBMP produced a 3.43 ± 0.75 µm hybrid layer in dentin prepared with diamond bur. This hybrid layer was regular and found consistently. In the air abrasion group, SBMP produced a 4.94 ± 1.28 µm hybrid layer, which was regular and found consistently. Statistical ANOVA (P = 0.05) indicated that there was a statistically significant difference between the groups. These data indicate that the air abrasion, within the parameters used in this study, provides a thick hybrid layer formation.