Plasma membrane (PM) association is an essential step in the retroviral life cycle that involves the complex association of the Gag polyprotein with cellular machinery, RNA, and specific lipid signatures of the inner leaflet. Here, I describe the first systematic comparison of Equine Infectious Anemia Virus (EIAV), Human Immunodeficiency Virus Type 1 (HIV-1), and Rous Sarcoma Virus (RSV) Gag membrane binding properties using in vitro liposome binding assays, in vivo virus release assays, and localization studies using confocal microscopy of 293T cells. By doing so, I hoped to address two main questions. First, what drives EIAV Gag association with the plasma membrane? And second, why is EIAV Gag not myristoylated? My results showed that EIAV Gag membrane association is driven by electrostatic interactions and a preference for cholesterol-containing membranes. These results highlight the universal importance of electrostatic interactions for retroviral Gag PM targeting and may underline a shared association with lipid raft microdomains for budding. I also used EIAV matrix (MA) and HIV-1 MA mutants to confirm HIV-1’s dependence on myristoylation for virus release and localization to the plasma membrane. These same mutants have also led me to propose a model for EIAV MA helix 1 inhibition of myristoylation. Finally, a part of my thesis work has been dedicated to the development of a novel silica-bead binding assay to study protein-lipid interactions. I describe this work in Appendix 1.