The adhesional contact between a particle and a substrate is a fundamental parameter for analyzing pull-off force data generated by atomic force microscopy (AFM). Roughness, present at some scale for all real materials, complicates this task by introducing asperity-con trot led contact. Roughness also causes pull-off force data scatter, a well-known phenomenon that is usually neglected in analysis of the AFM pull-off results. This paper presents the first systematic study of roughness effect on the pull-off force magnitude and its distribution characteristics. The results indicate that the scatter in the data decreases with increasing diameter of the probe as compared to the dimension of surface irregularities, but the magnitude of the pull-off force is more severely altered by roughness. The results also show that when particle size is at the same scale as surface roughness, multiple contact points may be made yielding increased adhesion.