Abstract: To analyze antigenic diversity of envelope protein among NDV strains isolated from different avian, monoclonal antibodies (MAbs) against WF00C strains of NDV isolated from chicken were prepared by whole virus which was purified by ultrafiltration. Then, the homogeneity and heterogeneity of MAbs were further analyzed by homology of antibody variable region and ownership of gene family, and key amino acid sites on variable epitope were determined by amino acid sequence analysis of antigenic site and reaction with expressed peptide, which were on the basis of its general biological characteristics and diversity comparison. Six MAbs were identified in this study, and named as 1B6, 4B4, 4E6, 4E11, 5H4 and 5H9. Except 4B4 and 5H4, the rest MAbs had hemagglutination inhibition (HI) activity, and neutralizing activity to NDV WF₀₀C strain. However, 1B6 and 4E11 which recognized linear epitopes didn’t react with two NDV strains isolated from pigeon in cross HI test. The homogeneity and heterogeneity analysis showed that antibody light and heavy chain variable region of 1B6 and 4E11, 4B4 and 5H4 belonged to the same gene family. 1B6 and 4E11 only had five and one amino acids difference in the light and heavy chain complementarity determining region, respectively, but 4B4 and 5H4 were identical. 4E6 and 5H9 had obvious difference in gene sources and amino acid sequences of complementarity determining regions. Amino acid sequence analysis of NDV HN antigenic site 14 showed that site of aa347 had six kinds of amino acids, where glycine was common amino acid for this site of NDV strains isolated from pigeon (26/27), and site of aa349 had three kinds of amino acids, where glutamate was specific to NDV strains isolated from pigeon. 1B6 and 4E11 recognized amino acid E347 and D349 which were variable sites and crucial for these epitopes. In this study, six MAbs against NDV which belonged to three groups according to biological characteristics could be attributed to four types on the basis of homology. Site of aa349 on HN protein being glutamate was specific to NDV strains isolated from pigeon, and this site was a new key amino acid site on the linear neutralizing epitope.