Novel duck reovirus (NDRV) causes high morbidity in ducklings, and recovered ducklings are often remarkably stunted in growth. In this study, four NDRV strains were isolated from the NDRV outbreaks that occurred in different regions of Shandong province, China. The biological characteristics and pathogenicity of the four NDRV strains were elucidated, and the N20 was identified as a naturally attenuated strain. Three-day-old ducklings were immunized with live N20 strain (100 ELD50/duck), and challenged with 104.52 ELD50 of virulent N19 strain at 7 days post immunization. The vaccinated ducklings showed no evidence of clinical signs, gross and histopathological lesions, or loss of body weight, and 100 % protection against the virulent NDRV N19 infection. The NDRV-specific antibodies were generated in the immunized ducklings and could neutralize different NDRV strains. These results indicated that the N20 strain was a promising live attenuated vaccine candidate against highly pathogenic NDRV infection.