Background: During early life, systemic protection to influenza is passively provided by transplacental transfer of IgG antibodies and oral and gastrointestinal mucosal protection via breast milk (BM) containing predominantly IgA. Immune imprinting, influenced by initial exposure of the infant immune system to influenza, has recently been recognized as an important determinant of future influenza immune responses. Methods: We utilized stored frozen BM from a prospective birth cohort to assess immune factors in human milk. The earliest available BM and a paired, timed serum sample was assessed from each of 7 mothers. Paired infant serum samples were assayed at up to three time points during the first 12 months of life, one prior to assumed disappearance of transplacentally transferred IgG, and one after. We utilized a novel multiplex assay to assess mothers’ and infants’ IgG and IgA antibodies in serum to a panel of 30 individual recombinant hemagglutinin (rHA) proteins of influenza virus strains and chimeric rHAs. We also characterized IgA and IgG antibody levels in breast milk which provide mucosal protection. Results: Our pilot results, analyzing a small number of samples demonstrate the feasibility of this method for studying paired maternal-infant IgG and IgA anti-influenza immunity patterns. Unlike IgG antibodies, breast milk influenza virus HA-specific IgA antibody levels and patterns were mostly discordant compared to serum. As expected, there was a steady decay of infant influenza specific IgG levels by 6 to 8 months of age, which was not, however, comparable in all infants. In contrast, most of the infants showed an increase in IgA responses throughout the first year of life Conclusions: This new analytical method can be applied in a larger study to understand the impact of maternal imprinting on influenza immunity.