Envelope glycoprotein (Env) of human immunodeficiency virus type 1 (HIV-1) is an important target for the development of an HIV vaccine. Extensive glycosylation of Env is an important feature that both protects the virus from antibody responses and serves as a target for some highly potent broadly neutralizing antibodies. Therefore, analysis of glycans on recombinant Env proteins is highly significant. Here we present glycosylation profiles of recombinant gp120 proteins from four major clades of HIV-1 (A, B, C, and AE) produced either as research-grade material in 293 and CHO cells or as two independent lots of clinical material under GMP conditions. Almost all potential N-linked glycosylation sites were at least partially occupied in all proteins. The occupancy rates were largely consistent among proteins produced under different conditions, although a few sites showed substantial variability even between two GMP lots. Our data confirmed previous studies in the field showing high abundance of oligomannose on Env protein, with 40-50% of glycans having Man5-Man9 on all four proteins under all production conditions. Overall the differences in occupancy and glycan forms among Env from different subtypes produced under different conditions were less dramatic than anticipated and antigenicity analysis with a panel of six monoclonal antibodies showed that all four gp120s maintained their antibody-binding profiles, including antibodies that recognize glycan forms. Such findings have major implications to the final production of a clinical HIV vaccine including Env glycoprotein components. IMPORTANCE HIV-1 Env protein is a major target for the development of an HIV-1 vaccine. Env is covered with a large number of sugar-based glycan forms - about 50% of the Env molecular weight is composed of glycans. Glycan analysis of recombinant Env proteins is important to understand its roles in vial pathogenesis and immune responses. The current report presents the first extensive comparison of glycosylation patterns of recombinant gp120 proteins from four major clades of HIV-1 produced in two different cell lines, grown at either laboratory condition or at 50L GMP scale across different lots. Information learned in this study is valuable for the further design and production of HIV-1 Env proteins as the critical components of HIV-1 vaccine formulations.