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Sugarcane mosaic virus (SCMV) (genus, Potyvirus; family, Potyviridae) is widespread, deleterious, and the most damaging pathogen of sugarcane (Saccharum officinarum L. and Saccharum spp.) that causes a substantial barrier to producing high sugarcane earnings. Sugarcane mosaic disease (SCMD) is caused by a single or compound infection of SCMV disseminated by several aphid vectors in a non-persistent manner. SCMV has flexuous filamentous particle of 700–750 nm long, which encapsidated in a positive-sense, single-stranded RNA molecule of 9575 nucleotides. RNA interference (RNAi)-mediated antiviral innate immunity is an evolutionarily conserved key biological process in eukaryotes and has evolved as an antiviral defense system to interfere with viral genomes for controlling infections in plants. The current study aims to analyze sugarcane (Saccharum officinarum L. and Saccharum spp.) locus-derived microRNAs (sof-miRNAs/ssp-miRNAs) with predicted potential for targeting the SCMV +ssRNA-encoded mRNAs, using a predictive approach that involves five algorithms. The ultimate goal of this research is to mobilize the in silico- predicted endogenous sof-miRNAs/ssp-miRNAs to experimentally trigger the catalytic RNAi pathway and generate sugarcane cultivars to evaluate the potential antiviral resistance surveillance ability and capacity for SCMV. Experimentally validated mature sugarcane (S. officinarum, 2n = 8X = 80) and (S. spp., 2n = 100–120) sof-miRNA/ssp-miRNA sequences (n = 28) were downloaded from the miRBase database and aligned with the SCMV genome (KY548506). Among the 28 targeted mature locus-derived sof-miRNAs/ssp-miRNAs evaluated, one sugarcane miRNA homolog, sof-miR159c, was identified to have a predicted miRNA binding site, at nucleotide position 3847 of the SCMV genome targeting CI ORF. To verify the accuracy of the target prediction accuracy and to determine whether the sugarcane sof-miRNA/ssp-miRNA could bind the predicted SCMV mRNA target(s), we constructed an integrated Circos plot. A genome-wide in silico-predicted miRNA-mediated target gene regulatory network was implicated to validate interactions necessary to warrant in vivo analysis. The current work provides valuable computational evidence for the generation of SCMV-resistant sugarcane cultivars.