Protein N-glycosylation is critical for many aspects of the biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the ongoing COVID-19 pandemic. The trimeric spike protein of SARS-CoV-2 mediates binding to and fusion with host cells, and is covered with a “glycan shield” of ~66 N-glycosylation sites per trimer. Viruses commonly gain N-glycosylation sites through evolution during ongoing circulation, and this can be associated with immune evasion by improved shielding of antigenic sites by N-glycans. This process can also in SARS-CoV-2, as evidenced by the Gamma variant, which has mutations that introduce two new N-glycosylation sequons compared to Wuhan-Hu-1. We expressed recombinant Wuhan-Hu-1 and Gamma spike from CHO cells and characterised their site-specific glycosylation with LC-MS/MS glycoproteomics, and antibody binding profiles. We found changes in site-specific glycosylation that could not have been predicted from bioinformatic analyses alone, and which caused changes in antibody binding profiles. Our results highlight the ongoing possibility of evolution of gain and loss of N-glycosylation sites in SARS-CoV-2 spike of relevance to viral infection and vaccine design.