Abstract Abs can acquire N-linked glycans in their V regions during Ag-specific B cell responses. Among others, these N-linked glycans can affect Ag binding and Ab stability. Elevated N-linked glycosylation has furthermore been associated with several B cell–associated pathologies. Basic knowledge about patterns of V region glycosylation at different stages of B cell development is scarce. The aim of the current study is to establish patterns of N-glycosylation sites in Ab V regions of naive and memory B cell subsets. We analyzed the distribution and acquisition of N-glycosylation sites within Ab V regions of peripheral blood and bone marrow B cells of 12 healthy individuals, eight myasthenia gravis patients, and six systemic lupus erythematosus patients, obtained by next-generation sequencing. N-glycosylation sites are clustered around CDRs and the DE loop for both H and L chains, with similar frequencies for healthy donors and patients. No evidence was found for an overall selection bias against acquiring an N-glycosylation site, except for the CDR3 of the H chain. Interestingly, both IgE and IgG4 subsets have a 2-fold higher propensity to acquire Fab glycans compared with IgG1 or IgA. When expressed as rmAb, 35 out of 38 (92%) nongermline N-glycosylation sites became occupied. These results point toward a differential selection pressure of N-glycosylation site acquisition during affinity maturation of B cells, which depends on the location within the V region and is isotype and subclass dependent. Elevated Fab glycosylation represents an additional hallmark of TH2-like IgG4/IgE responses.