The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a critical global health challenge, with many molecular pathogenism mechanisms remaining unclear. Among small noncoding RNAs, tRNA-derived RNA fragments (tRFs) have gained attention for their roles in viral infections. We previously reported significant alterations in tRFs originating from the 5’-end of tRNAs (tRF5s) in nasopharyngeal swab (NPS) specimens from COVID-19 patients infected with the SARS-CoV-2 alpha variant. Building upon this work, we collected 209 NPS specimens between late 2021 and February 2023. This cohort of specimens includes patients positive for delta or omicron variants (BA.1, BA.5, XBB.1.5), and negative controls. We investigated five tRF5s (tRF5-GlnCTG, tRF5-CysGCA, tRF5-ValCAC, tRF5-GlyCCC2, and tRF5-GlyGCC) expression among this cohort and observed distinct tRF5s induction patterns among the different viral variants. tRF5-ValCAC, which we previously reported was induced by the alpha variant, was absent among later variants. tRF5-GlnCTG was consistently elevated by multiple variants (alpha, delta, and BA.1), while the induction diminished in BA.5 and XBB.1.5. tRF5-GlyCCC2 induction had not been observed until the BA.1 wave; its induction initially occurred in the pediatric population with BA.1 or BA.5, and later within both pediatric and adult populations with XBB.1.5. Subsequently, cytokines/chemokines within these NPS specimens were measured via Bio-plex. We found several tRF5s levels correlated with cytokines/chemokines. Functional study results suggested these tRF5s regulate the replication of their corresponding SARS-CoV-2 variants. Our findings highlight a potential link between tRF5s and disease severity, offering insights into their functional roles in viral replication and their potential as biomarkers.