Virus filtration is a downstream unit operation in the manufacturing of biotherapeutics to remove potential viral contaminations based on size exclusion. While even very small viruses are effectively retained under normal flow conditions, process interruptions can compromise the virus-reduction capacity of a filter. Yet, direct insights into the underlying flow-dependent retention and breakthrough mechanisms of relevant parvoviruses are still lacking. To study the retention of parvoviruses inside the polymeric structures of four commonly used filter types, minute virus of mice (MVM) was fluorescently labeled and visualized in membrane cross-sections post-filtration by laser scanning microscopy. The virus retention profiles revealed a membrane structure-specific accumulation of viral particles at a distinct depth in the separation-active layers. Pressure release experiments showed that flow interruption-induced virus breakthrough is associated with the mobilization and deeper migration of viruses into denser membrane layers. Moreover, we discovered that local clusters of breakthrough foci in a particular filter type are responsible for the substantial transmission of viruses to the filtrate. Taken together, the membrane-specific phenomena visualized herein contribute to a better understanding of the underlying virus retention mechanisms and provide cues for a specific optimization of virus filtration processes. Display omitted •First comparative visualization of a relevant parvovirus in different filters.•Viruses are retained in characteristic membrane structure-related patterns.•Flow interruptions lead to mobilization and deeper migration of viruses.•Clusters of breakthrough foci cause substantial virus penetration through filters.