Retroviruses such as the human immunodeficiency virus, human T-cell lymphotropic virus and murine leukaemia virus are believed to spread via sites of cell-cell contact designated virological synapses. Support for this model is based on in vitro evidence in which infected cells are observed to specifically establish long-lived cell-cell contact with uninfected cells. Whether virological synapses exist in vivo is unknown. Here we apply intravital microscopy to identify a subpopulation of B cells infected with the Friend murine leukaemia virus that form virological synapses with uninfected leucocytes in the lymph node of living mice. In vivo virological synapses are, like their in vitro counterpart, dependent on the expression of the viral envelope glycoprotein and are characterized by a prolonged polarization of viral capsid to the cell-cell interface. Our results validate the concept of virological synapses and introduce intravital imaging as a tool to visualize retroviral spreading directly in living mice.