Using the Hemagglutinin (HA) protein peptide array of H1N1 pdm09 and a panel of swine antisera against various swine influenza H1 and H3 clusters, we identified three immunoreactive epitopes with one (peptide 15) located in HA1 (amino acids 57-71) and two (peptides 121 and 139) in HA2 (amino acids 481-495 and 553-566). Further analysis showed that all swine antisera of H1 clusters efficiently recognized two HA2 epitopes; peptides 121 and 139, with only a subset of antisera reactive to HA1-derived peptide 15. Interestingly, none of these peptides were reactive to SIV H3 antisera. Finally, intranasal inoculation of peptides 15 and 121 into pigs revealed that peptide 121, not peptide 15, was able to generate antibody responses in some animals. The results of our experiments provide an important foundation for further analyzing the immune response against these peptides during natural viral infection and also provide peptide substrates for diagnostic assays.