We have identified a gene, lpt‐3, that is required for the addition of phosphoethanolamine to the 3‐position (PEtn‐3) on the β‐chain heptose (HepII) of the inner core lipopolysaccharide (LPS) of Neisseria meningitidis (Nm). The presence of this PEtn‐3 substituent is characteristic of the LPS of a majority (≈ 70%) of hypervirulent Nm strains, irrespective of capsular serogroup, and is required for the binding of a previously described monoclonal antibody (mAb B5) to a surface‐accessible epitope. All strains of Nm that have PEtn‐3 possess the lpt‐3 gene. In some lpt‐3‐containing strains, the 3‐position on HepII is preferentially substituted by glucose instead of PEtn, the result of lgtG phase variation mediated by slippage of a homopolymeric tract of cytidines. Inactivation of lpt‐3 resulted in loss of PEtn‐3, lack of reactivity with mAb B5 and conferred relative resistance to bactericidal killing and opsonophagocytosis by mAb B5 in vitro. Thus, the identification of lpt‐3 has facilitated rigorous genetic, structural and immunobiological definition of an immunodominant epitope that is a candidate immunogen for inclusion in an LPS‐based vaccine to protect against invasive meningococcal disease.