Human parechoviruses (HPeVs), belonging to the Picornaviridae family, are clinically relevant human pathogens. Based on nucleotide sequence diversity, HPeVs are classified into 17 genotypes of which HPeV3 in particular has been associated with severe disease in young infants. HPeV3 is frequently detected in clinical specimens worldwide and several outbreaks have been described, most recently in Japan and Australia. However, the observed seropositivity rates for HPeV3 neutralizing antibodies (nAbs) vary from high in Japan to low in the Netherlands and Finland. To study if antigenic diversity among HPeV3 strains included in the serological studies explains the observed variation, we determined the neutralizing activity of Japanese and Dutch intravenous immunoglobulin batches (IVIG), a rabbit HPeV3 hyperimmune polyclonal serum, and a human HPeV3-specific monoclonal antibody (mAb) AT12-015, against the HPeV3 A308/99 prototype strain and clinical isolates from Japan, the Netherlands and Australia, collected between 1989 and 2015. The rabbit antiserum could neutralize all HPeV3 isolates whereas the neutralization capacity of the IVIG batches varied, and the mAb exclusively neutralized the A308/99 strain. Mapping of the amino acid variation among a subset of the HPeV3 strains on an HPeV3 capsid structure revealed the VP1 C-terminus as a likely immunogenic region. Furthermore, four amino acid mutations in a mAb AT12-015-resistant HPeV3 A308/99 variant indicated the location for potential antigenic determinants. The observed antigenic diversity in HPeV3 can explain the varying levels of nAb seropositivity and facilitate persistent circulation and outbreaks. Seroepidemiological study of nAbs against the Australian 2013 HPeV3 outbreak strain in Australian, European and North-American general population is ongoing.