Significance The HIV-1 Env “glycan shield” masks the surface of the protein from immune recognition, yet intrinsic heterogeneity defies a typical structure–function description. Using an integrated approach of cryo-EM, computational modeling, and mass spectrometry, we visualized the glycan shield structure in a new light. Our approach facilitated development of cryo-EM analysis methods and allowed for validation of models against experiment. Comparison of Env expressed in different cell lines revealed how subtle differences in composition impact glycan shield structure and affect the accessibility of epitopes on the surface, providing insights for vaccine design. Finally, time-resolved cryo-EM experiments uncovered how highly connected glycan clusters help stabilize the prefusion trimer, suggesting the glycan shield may function beyond immune evasion.