Atomic force microscopy (AFM) and positron annihilation spectroscopy (PAS) were applied to study structure of filler agglomerates in rubber matrix. Mixes of silica and ethylene-propylene-diene rubber (EPDM) were prepared either on a two-roll mill or in internal laboratory mixer and thus subjected to vulcanization. Both rubber mixes as well as vulcanizates before and after intensive wear treatment were studied. Apart influence of a macro-scale filler distribution, claimed in the subject literature, significant differences in their micro-scale distribution as well as internal structure of aggregates/agglomerates were also detected, that are likely also to play a role in exploitation performance of rubber. It was especially visible for the two roll mill mixes, prepared under the most intensive mixing conditions (high-loaded pre-mixes technology) resulting in filler agglomeration, whereas for the internal mixer samples mainly non-uniform filler distribution, without agglomeration, took place. Despite similar value of the dispersive index (DI)-ISO 11345, rubber samples can exhibit different mechanical and tribological properties.