The USDA-APHIS Plant Germplasm Quarantine Program (PGQP) is the first line of defense against the entry, establishment and spread of quarantine pathogens with the potential to damage the U.S. economy, food production and natural resources. The number and type of prohibited plants requiring quarantine is increasing, and with it, the need for implementing new technologies that will more rapidly detect a wider range of high priority pathogens. High-throughput sequencing (HTS) technologies are being adopted worldwide as a powerful diagnostic tool, requiring no prior knowledge of the pathogens infecting the plant material. To validate the use of HTS as a diagnostic tool in the PGQP, we assessed whether HTS can consistently identify 10 regulatory-significant viruses across time, and the sequencing depth needed for their identification. Total RNA from plant samples was extracted in spring and fall and independently sequenced using RNA shotgun metagenomics. Our results showed that: 1) HTS can consistently identify the same DNA and RNA viruses across seasons; 2) the percentage of viral reads identified per sample is higher for RNA than DNA viruses, ranging from as low as 3.14E-03% to 45%; 3) indexing of Poaceae germplasm by HTS is recommended to be performed during the spring season; and 4) the sequencing depth needed for RNA virus identification is 0.5M reads vs. 25M for DNA viruses. Collectively, findings from this work are providing a solid foundation for the use of HTS as a diagnostic tool in a regulatory setting, such as for the processing of quarantined germplasm.