Duck enteritis virus (DEV) causes an acute and contagious infection in duck. The present study was carried out to evaluate the pathogenicity and pathodynamics of DEV isolates from different natural outbreaks in the Assam Province of India. A total of six wild-type isolates of DEV were revived in ducklings to determine its biologic characterization. Postmortem examination of infected ducklings revealed DEV-specific gross lesions in different organs. The presence of DEV was confirmed by its genome amplification and the presence of viral antigens from collected tissue samples by indirect fluorescent antibody test. All the isolates revived in ducklings were further propagated in duck embryo fibroblast cells. Highly virulent and low virulent isolates of DEV were selected for further study based on median duck infectivity dose (DID50) and median tissue culture infectivity dose (TCID50). The highly virulent isolate of DEV had values of 102 DID50/ml and 106.33 TCID50/ml, whereas the low virulent strain had titers of 10 DID50/ml and 104.83 TCID50/ml in the cell culture. Our results showed replication of DEV in ducks with the highest and lowest viral titers in the thymus and bursa of Fabricius, respectively. In addition, microscopic analysis revealed necrosis and degeneration of submucosal esophageal glands and glandular epithelium. The study will be useful to understand the organ tropism and pathologic alteration among the virulent DEV isolates.