Abstract: To investigate the genetic variation and evolutionary patterns of genotype II grass carp reovirus (GCRV-II), in this study, we analyzed 20 GCRV-II isolates collected from Guangdong, Hunan, Jiangxi, and other provinces from 2017 to 2025. Third-generation single-molecule nanopore sequencing was employed to perform single nucleotide variant (SNV) analysis on the S10 and S11 genome segments of GCRV-II. The number and density of variant sites, together with the Shannon entropy diversity index, were used to evaluate differences in quasispecies genetic diversity among different GCRV-II strains. The results showed that varying degrees of nucleotide variation occurred in both the S10 and S11segments of the 20 GCRV-II isolates, with each variant site containing at least two nucleotides and nucleotide transitions predominating. Overall, the S10segment exhibited a higher level of variation than the S11segment. SNV density ranged from 0 to 144.1 SNVs/kb in the S10segment and from 0 to 21.4 SNVs/kb in the S11segment. Shannon entropy analysis revealed marked differences in quasispecies genetic diversity among GCRV-II isolates collected at different times and from different geographical regions, suggesting that quasispecies variation among GCRV-II strains may be associated with geographic origin or epidemic period. Phylogenetic analysis further demonstrated clear evolutionary divergence among the 20 GCRV-II isolates. Based on third-generation single-molecule nanopore sequencing, this study compared quasispecies genetic diversity at the single-nucleotide level in the S10 and S11segments of GCRV-II strains from different sampling years and geographical origins. These findings provide a foundation for further elucidating the genetic variation patterns and evolutionary mechanisms of GCRV-II, and offer theoretical support for vaccine candidate selection and epidemiological surveillance.