Clonal grasses imported into Australia for use as nursery stock are permitted as bare-rooted grasses or tissue culture plantlets and should complete a minimum 6-month quarantine period at Australia’s Post Entry Quarantine Facility (PEQ). During this period, established plants are tested for plant pathogens using a variety of tools. Viral diagnostics in clonal grasses are reliant on visual inspection and small RNA sequencing (sRNAseq). All viral detections are assessed for the risk case-by-case due to the large number of species imported and limited literature available on viruses in clonal grasses in Australia. Since 2022, 95 clonal grass extracts from 7 species have been tested through sRNAseq. Successful viral detection in sRNASeq is heavily reliant on the quality of the RNA extract and the enrichment of miRNAs and minimalisation of plant RNAs during the library preparation. After bioinformatics processing of the raw reads for quality followed by removal of plant reads and size filtering for 21-22nt reads, at least 4 million informative reads are retained per sample for de novo assembly. On several occasions, clonal grass samples failed to retain adequate numbers of reads that were informative for downstream analysis. This resulted in re-extracting and/or resequencing samples for diagnostics and in some cases, an extended quarantine period for these plants. In those problematic samples, a higher percentage of reads corresponding to fragments of plant tRNA was found, likely preventing the retention of adequate informative reads in those samples. Homology searches of overrepresented sequences from individual samples revealed that samples from the same species were dominated by distinct types of tRNAs. A high percentage of plant reads such as tRNA in the sequenced read pool can reduce the overall sensitivity of our diagnostic approach which is targeting the viral-derived small RNAs. Therefore, we compared a new library preparation kit with the current method for their efficacy in filtering the tRNA. Although the current protocol underperformed in filtering tRNA, it was found to have a higher overall sensitivity than the new kit. We also compared the tissue types of leaf blade vs. stem/internode of Arundo donax and Stenotaphrum secundatum for their tRNA content and overall performance in pathogen detection. This study revealed that the tRNA content in the internode is significantly less than in the leaf blade extracts of the same plant for both grass species. Therefore, we recommend that internode is better suited for sampling for viral diagnostics in those two species. We aim to expand this investigation to other grass species to further improve the sensitivity of sRNAseq towards viral detection in clonal grasses.