The MA (matrix) domain of the retroviral Gag polyprotein plays several critical roles during virus assembly. Although best known for targeting the Gag polyprotein to the inner leaflet of the plasma membrane for virus budding, recent studies have revealed that MA also contributes to selective packaging of the genomic RNA (gRNA) into virions. In this Review, we summarize recent progress in understanding how MA participates in genome incorporation. We compare the mechanisms by which the MA domains of different retroviral Gag proteins influence gRNA packaging, highlighting variations and similarities in how MA directs the subcellular trafficking of Gag, interacts with host factors and binds to nucleic acids. A deeper understanding of how MA participates in these diverse functions at different stages in the virus assembly pathway will require more detailed information about the structure of the MA domain within the full-length Gag polyprotein. In particular, it will be necessary to understand the structural basis of the interaction of MA with gRNA, host transport factors and membrane phospholipids. A better appreciation of the multiple roles MA plays in genome packaging and Gag localization might guide the development of novel antiviral strategies in the future.