Cereals are annual common grasses in the Poaceae family, formerly known as the Gramineae. Grains are the most popular cereal product and account for more than 50% of all calories consumed globally. Grain crop yields are at risk from disease infections; in severe cases, production might decrease by 80%. Therefore, the agricultural biosecurity system in the United States has put in place biosecurity measures to restrict and reduce the financial impact of pathogen infections represented by the USDA-APHIS- Plant Germplasm Quarantine Program (PGQP). Rapid diagnostics methods are required for disease surveillance and monitoring to avoid pathogen spread. In this regard, High Throughput Sequencing (HTS) holds great promise for the simultaneous detection and surveillance of many possible plant pathogens. Microbe Finder (MiFi®) is a graphical user interface for bioinformatics designed to fill the gap between pathogen detection and HTS. The objectives of this study are to develop a novel diagnostic tool capable of detecting the 31 Poaceae viruses restricted by the USDA-APHIS-PGQP and to describe and characterize two new viral genomes infecting Miscanthus sinensis leaf tissue described on HTS metagenomic sequencing. E-probes were designed, curated, and tested in silico using a metagenome spiked with the virus sequence. The theoretical limit of detection was assessed for three of those viruses using in silico predictive models. In vitro validation was conducted with nine viruses from the sugarcane plant HTS sequenced by Illumina and MinION to compare different methods of sequence and tested on MiFi®. New PCR primers for the 31 viruses were also designed for confirmation, and sequencing obtained by both outputs were assessed by mapping to reference. For the second objective new virus sequences were analyzed and annotated using Geneious Prime v.2021.2.2 and the complete genome sequence was reported to ICTV. Primers were designed for PCR amplification and confirmation. Sanger sequence and biological assays with 22 different species of plants were conducted to observe the transmissibility and reliability of the HTS sequence. Finally, a phylogenetic analysis was performed to reveal the family and genus the two new viruses are grouped.