Surveys were conducted in barley (Hordium vulgare L.) fields of Khorasan Razavi Province, Northeast Iran, during 2015-2017. A total of 120 soil and root samples were collected to examine the prevalence of Plant parasitic nematodes. In morphological and morphometrical identification 18 species were recorded belonging to nine genera. Among these species, Rotylenchus alius was found as a new record from Iran and Basiria gracilis, Boleodorus thylactus, Ditylenchus apus and Meloidogyne arenaria were reported as new host record from barley fields of Iran. Meloidogyne arenaria, was selected for detailed investigation at molecular level. Molecular traits on Meloidogyne arenaria species done by partial 18s ribosomal DNA primer and sequence results verified the morphometric studies and showed 99 percent resemblance to AB905316 sequence from Japan. In addition, scanning electron microscope (SEM) assay on infected roots by Meloidogyne arenaria showed that this species has been able to create gall in barley root and disrupt on cellular metabolism.