Monitoring avian influenza (AI) infection and detecting silent infection in vaccinated chickens has been challenging due to the lack of effective serological diagnostic assays to differentiate between vaccinated and infected animals. Very few studies have identified suitable proteins in AI virus that can be used in successfully differentiating infected from vaccinated animals (DIVA). An HA2 peptide: HA2 position 197-201 (HA position 488-516) described by Khurana et al. (J Virol 85(23):12455-12463, 2011), was shown to have DIVA ability by differentiating H5N1-infected human sera in ELISA. In order to explore the capacity of the HA2 protein, as a DIVA reagent in chickens, four overlapping recombinant HA2 proteins, were expressed in E. coli and tested for reaction with H5N1 sera obtained from infected and vaccinated chickens. Recombinant protein HA2_B2 (380-461) was able to generate a detectable reaction with both H5N1 infected and vaccinated chicken sera but recombinant protein HA2_B4 (483-565) reacted strongly only with sera obtained from chickens infected with live virus, confirming its suitability as a DIVA antigen. Further analysis of the HA2 using several overlapping peptides suggested that positions 380-461 and 483-565 were antigenic in mouse and chicken. This study, for the first time, identified novel antigenic epitopes on the H5N1 HA2 subunit. Two epitopes, found in the HA2 ectodomain, have never been described for AIV infection in any animal species. Also one HA2 epitope was found to have high potential as a DIVA antigen.