Cannabis sativa (hemp) is currently undergoing a rediscovery in different economically relevant fields, from industrial to medical, leading to an increase of cultivated areas. Virome of this plant has been only recently explored through NGS technologies, leading to detection of known and unknown viruses (1). Cryptic viruses, characterized by persistent infection without symptoms, have been isolated from many plants including hemp. Cannabis cryptic virus (CanCV, family Partitiviridae) and Cannabis sativa mitovirus 1 (CasaMV1, family Mitoviridae) have been reported as widely distributed in C. sativa (2,3). In this work, we performed an Illumina small RNA (sRNA) sequencing analysis of different varieties of C. sativa to reconstruct their virome components and characterize antiviral defenses based on RNA interference (RNAi). Total RNA was extracted from 15 dioecious plants cultivated for cannabinoid production (CBD or CBG) and 4 monoecious plants cultivated for seeds and fiber production. De novo and reference-based assembly of sRNA reads allowed us to detect both CanCV and CasaMV1 and reconstruct their complete genome sequences. Incidence of CanCV was lower than expected (2 of 19 plants) and this virus was targeted by antiviral RNAi generating predominantly 21 and 22 nt small interfering RNA (vsiRNAs) from both strands of the viral genome, suggesting that their biogenesis is mediated by Dicer-like (DCL) 4 and DCL2 enzymes, respectively. CasaMV1 had a higher incidence (42.1% plants) and its vsiRNAs had a wider size range and a positive strand bias, with 21 nt vsiRNAs being the most represented on both strands. Analysis of 5'-terminal nucleotide identity of sRNAs indicated that the major size classes of vsiRNAs derived from both viruses are mainly associated with Argonaute (AGO) 1-like (5'U) and AGO5-like (5'C) enzymes that can catalyze cleavage and degradation of viral RNA.