It was shown earlier that the reassortant influenza virus having hemagglutinin (HA) gene of A/Duck/Primorie/2621/2001 (H5N2) virus and 7 genes of A/Puerto Rico/8/34 (H1N1) virus produced low yields in embryonated chicken eggs. We found that a variant reassortant selected by serial passages in eggs produced higher yields than the initial reassortant. The variant reassortant had an amino acid substitution in the hemagglutinin N244D (H3 numbering). In this report we demonstrated that the post-reassortment amino acid substitution N244D altered the antigenic specificity of HA as revealed by the loss of reactivity with an anti-H5 monoclonal antibody in hemagglutination-inhibition (HI) test. The results are discussed in association with the evolution of H5 hemagglutinin.