design: The diversity of viruses in the Yangtze River remains largely unexplored. Toinvestigatethefreshwater viromefrom the Yangtze River, totally thirty litres of water samples were collected from six river ports along the Yangtze River in 2017. Eachsamplewascondensed into one milliliterandvigorouslyvortexedfor5min. Thesupernatantswerethencollectedaftercentrifugation.500ulofeachsupernatantwasfilteredthrougha0.45umfiltertoremoveeukaryoticandbacterialcellsizedparticles.ThefiltratesenrichedinviralparticlesweretreatedwithDNaseandRNasetodigestunprotectednucleicacidfor60min.RemainingtotalnucleicacidwasthenisolatedusingQIAampMinEluteVirusSpinKitaccordingtomanufacturerprotocol.SixlibrarieswerethenconstructedusingNexteraXTDNASamplePreparationKitandsequencedusingtheIlluminaMiSeqplatform with250basespairedendswithdualbarcodingforeachlibrary.Forbioinformaticsanalysis, pairedendreadsof250bpgeneratedby IlluminaMiSeqplatform  weredebarcodedusingvendorsoftwarefromIllumina.