Hemp (Cannabis spp.) is a rich source of natural compounds and fiber for medicinal and industrial use. While fungal and bacterial pathogens of hemp are quite well characterized, virus infections have been reported and seen as an emerging threat for hemp cultivation only in recent years. In this work we used Illumina small RNA sequencing for virome reconstruction and characterization of the antiviral defense based on RNA interference (RNAi) in industrial hemp plants (monoecious) and dioecious plants cultivated for production of CBD/CBG cannabinoids. By de novo and reference-based assembly of small RNA reads we identified and reconstructed previously-reported viruses such as Cannabis cryptic virus (family Partitiviridae), Cannabis sativa mitovirus 1 (Mitoviridae) and Grapevine line pattern virus (Bromoviridae) as well as a putative new species of Partitiviridae. Members of both Partitiviridae and Bromoviridae families were targeted by antiviral RNAi generating predominantly 21 and 22 nt small interfering RNAs from both strands of the entire virus genome. In contrast, mitovirus-derived small RNAs belonged to a wider size range, with 16 and 21 nt size classes being the most abundant, and resembled Cannabis mitochondrion genome-derived small RNAs. Association of disease symptoms (if any) with identified viruses and/or relative abundance of viral sRNAs will be presented. To our knowledge, this is the first characterization of antiviral RNAi in hemp plants.