Significance Effective prevention strategies are urgently needed to control the spread of HIV-1. A critical barrier to developing such strategies is the lack of understanding of the host antiviral defenses that control HIV-1 replication in the mucosa at the site of entry. Here, we characterized viruses from matched donor and recipient pairs to determine whether transmitted HIV-1 strains exhibit traits that increase their transmission fitness. Characterizing 300 limiting dilution-derived isolates, we identified several properties that enhance virus replication in the face of a vigorous innate immune response, of which resistance to type 1 IFNs is the most important. These results provide new insights into the HIV-1 transmission process and define possible new targets for AIDS prevention and therapy.