Dimerization of the RNA genome is a key event in HIV-1 virion assembly and has a strong impact in viral replication and evolution. Packaging the dimeric genome allows frequent recombination to rescue genetic information in damaged RNAs and to generate variants that can evade the host immune response or resist antiviral treatments. Furthermore, genome packaging is regulated by recognition of dimeric RNA. Our studies demonstrate that HIV-1 RNAs dimerize not in the cytoplasm but on the plasma membrane, often early during the assembly process, and that Gag protein is required for maintenance of the RNA dimer. These studies address the timing, location, and partners involved in RNA dimerization, an important process for HIV-1 replication.