Prior to each annual flu season, health authorities recommend three or four virus strains for inclusion in the annual influenza vaccine: a Type A:H1N1, a Type A:H3N2, and one or two Type B viruses. Antigenic differences between strains are found in the glycosylation patterns of the major influenza antigen, hemagglutinin (HA). Here we examine the glycosylation patterns of seven reference antigens containing HA used in influenza vaccine potency testing. These reagents are supplied by the Center for Biologics Evaluation and Research (CBER) or the National Institute for Biological Standards and Control (NIBSC) for use in vaccine testing. Those produced in hen egg, Madin Darby Canine Kidney (MDCK), and insect (Sf9) expression systems were examined. They are closely related or identical to antigens used in commercial vaccine. Reference antigens studied were used in the 2014-2015 influenza season and included A/California/07/2009 H1N1, A/Texas/50/2012 H3N2 and B/Massachusetts/02/2012. Released glycan and HA specific glycopeptide glycosylation patterns were examined. Also examined was the sensitivity of the Single Radial Immunodiffusion Assay (SRID) potency test to differences in HA antigen glycosylation. The SRID assay was not sensitive to any HA antigen glycosylation status from any cell system based on deglycosylation studies as applied using standard assay procedures. Mapping of glycosites with their occupying glycan to functional regions, including antigenic sites, lectin interaction regions and fusion domains was performed and has implications for immune processing, immune response and antigenic shielding. Differences in glycosylation patterns, as dictated by cell system used in expression, may impact on these functions.IMPORTANCE Here the glycosylation patterns of the 2014-2015 influenza vaccine season standard antigens A/California/07/2009 H1N1, A/Texas/50/2012 H3N2, and B/Massachusetts/02/2012 were revealed and sensitivity of the Single Radial Immunodiffusion Assay (SRID) potency test glycosylation was tested. Differences in hemagglutinin glycosylation site composition and heterogeneity seen in antigen produced in different cell substrates suggests differences in processing and downstream immune response. The SRID potency test used in vaccine release, is not sensitive to differences in glycosylation when applied under standard use conditions. This work reveals important differences in vaccine antigens and may point toward areas where improvements may be made concerning vaccine antigen preparation, immune processing and testing.