Abstract: A new grass carp virus, assigned JX09-01, was isolated from a diseased grass carp case during routine examination in Nanchang City, Jiangxi Province. The obvious cytopathic effects (CPE) were observed after the new isolate inoculated in grass carp kidney cell (CIK), grass carp liver cell (L8824) and proboscis fibroblast cell (PSF) respectively, and plenty of non-enveloped virus particles with hexagonal shape were observed under electron microscope (EM), which was highly similar to reported grass carp reovirus (GCRV). The specific bands could be detected by reverse transcription-polymerase chain reaction (RT-PCR) from infected fish and cells using primer designed according to the sequences of GCRV 873 strain, but no result obtained using primers designed with reference to the sequence of GCRV HZ08 strain. Sequence analysis of segment 6 showed that the nucleotide sequence similarity between JX09-01 and other GCRV strains was 99.3% (GCRV 873) and 30.4% (GCRV HZ08), while the deduced amino acid sequence similarity were 98.6% and 30% respectively, which indicated the JX09-01 is grass carp reovirus. The pathogenicity test on grass carp found that new separated virus was a mild strain. The grass carps with 8-12 cm length were immunized with the new isolate and protection rate of 86.7% was obtained against virulent strain challenge, which indicated that the new separated strain could be a candidate for attenuate vaccine strain as a grass carp reovirus.