Immune responses against certain viruses are accompanied by autoantibody production although the origin of these infection-associated autoantibodies is unclear. Here we report that murine gamma-herpesvirus 68 (MHV68)-induced autoantibodies are derived from polyreactive B cells in germinal center (GC) through the activity of short-lived plasmablasts. The analysis of recombinant antibodies from MHV68-infected mice revealed that about 40% of IgG+ GC B cells were self-reactive, with about half of them being polyreactive. On the other hand, virion-reactive clones accounted for only a minor proportion of IgG+ GC B cells, half of which also reacted with self-antigens. The self-reactivity of most polyreactive clones was dependent on somatic hypermutation (SHM), but this was dispensable for the reactivity of virus mono-specific clones. Furthermore, both virus-mono-specific and polyreactive clones were selected to differentiate to B220lo CD138+ plasma cells (PCs). However, the representation of GC-derived polyreactive clones was reduced and that of virus-mono-specific clones was markedly increased in terminally differentiated PCs as compared to transient plasmablasts. Collectively, our findings demonstrate that, during acute MHV68 infection, self-reactive B cells are generated through SHM and selected for further differentiation to short-lived plasmablasts but not terminally differentiated PCs.