Wheat streak mosaic virus (WSMV) is a persistent threat to wheat production, necessitating novel approaches for protection. We developed an artificial miRNA strategy against WSMV,incorporating five amiRNAs within one polycistronic amiRNA precursor. Using miRNAsequence and folding rules, we chose five amiRNAs targeting conserved regions of WSMVbut avoiding off-targets in wheat. These replaced the natural miRNA in each of five arms ofthe polycistronic rice miR395, producing amiRNA precursor, FanGuard (FGmiR395), whichwas transformed into wheat behind a constitutive promoter. Splinted ligation detected all fiveamiRNAs being processed in transgenic leaves. Resistance was assessed over two generations.Three types of response were observed in T1 plants of different transgenic families: completelyimmune; initially resistant with resistance breaking down over time; and initially susceptiblefollowed by plant recovery. Deep sequencing of small RNAs from inoculated leaves allowedthe virus sequence to be assembled from an immune transgenic, susceptible transgenic, andsusceptible non-transgenic plant; the amiRNA targets were fully conserved in all three isolates,indicating virus replication on some transgenics was not a result of mutational escape by thevirus. For resistant families, the resistance segregated with the transgene. Analysis in the T2generation confirmed the inheritance of immunity and gave further insights into the otherphenotypes. Stable resistant lines developed no symptoms and no virus by ELISA; this resistancewas classified as immunity when extracts failed to transmit from inoculated leaves totest plants. This study demonstrates the utility of a polycistronic amiRNA strategy in wheatagainst WSMV.