Canine distemper virus (CDV) is an economically important virus responsible for canine distemper (CD), a highly contagious disease that afflicts various animal species worldwide. The hemagglutinin (H) protein is the major neutralizing target of virus. Therefore, it is often considered as immunogen to prepare neutralizing antibodies. The accurate identification of neutralizing epitope will provide important antigenic information and extend the knowledge of mechanisms of virus neutralization. In this study, we generated a neutralizing monoclonal antibody (mAb) 4C6 against CDV H protein, and defined the minimal linear epitope 238DIEREFDT245, which was highly conserved in America-1 genotype of CDV strains (vaccines). The mAb 4C6 could not react with a CDV strain that had two substitutions of D238Y and R241G in the epitope, which appeared in most CDV strains of the other genotypes. Besides, a few different amino acid mutations in the epitope were also included. Collectively, the epitope 238DIEREFDT245 was variable in the other genotypes of CDV strains. The epitope 238DIEREFDT245 was exposed to the surface of CDV H protein, showing good antigenicity. These data will provide insights into structure, function and antigenicity of H protein and lay the foundation for the development of diagnostic technologies and vaccine design for CDV.