Glioblastomas (GBMs) are the most lethal brain cancer with a median survival rate of fewer than 15 months. Both clinical and biological features of GBMs are largely different from those of low-grade gliomas (LGs), but the reasons for this intratumoral heterogeneity are not entirely clear. Transfer RNA (tRNA)-derived small RNAs (tsRNAs) were derived from tRNA precursors and mature tRNA, referring to the specific cleavage of tRNAs by dicer and angiogenin (ANG) in particular cells or tissues or under certain conditions such as stress and hypoxia. With the characteristics of wide expression and high stability, tsRNAs could be used as favorable biomarkers for diagnosis, treatment, and prognosis prediction of the tumor, viral infection, neurological as well as other systemic diseases. In this study, we have compared the differential expressed tsRNAs between GBMs and LGs, so as to investigate the possible pathogenic molecules and provide references for discovering novel nucleic acid drugs in future studies., Fresh tumor tissues of patients that were diagnosed as GBMs (4 cases) and LGs (5 cases) at the First Affiliated Hospital of Wenzhou Medical University from 2019.05 to 2021.01 were collected. The tsRNAs' levels were analyzed and compared through high-throughput sequencing, candidate tsRNAs were chosen according to the expression level, and the expression of the candidate tsRNAs was validated through qPCR. Finally, the potential targets were imputed using the Miranda and TargetScan databases, and possible biological functions of the differentially expressed (DE) tsRNAs' targets were enriched based on GO and KEGG databases., A total of 4 GBMs and 5 LGs patients were enrolled in the current study. High-throughput sequencing showed that 186 tsRNAs were expressed in two groups, over them, 43 tsRNAs were unique to GBMs, and 24 tsRNAs were unique to LGs. A total of 9 tsRNAs were selected as candidate tsRNAs according to the tsRNA expression level, among which 6 tsRNAs were highly expressed in GBMs and 3 tsRNAs were low expressed in GBMs. qPCR verification further demonstrated that 5 tsRNAs were significantly up-regulated and 1 tsRNA was significantly down-regulated in GBMs: tRF-1-32-chrM.Lys-TTT (p=0.00118), tiRNA-1-33-Gly-GCC-1 (p=0.00203), tiRNA-1-33-Gly-CCC-1 (p=0.00460), tRF-1-31-His-GTG-1 (p=0.00819), tiRNA-1-33-Gly-GCC-2-M3 (p=0.01032), and tiRNA-1-34-Lys-CTT-1-M2 (p=0.03569). Enrichment analysis of the qPCR verified DE tsRNAs showed that the 5 up-regulated tsRNAs seemed to be associated with axon guidance, pluripotent stem cells regulation, nucleotide excision repair, Hippo signaling pathway, and cancer-related pathways, while the down-regulated tsRNA (tRF-1-32-chrM.Lys-TTT) was associated with oocyte meiosis and renin secretion., The tsRNAs were differentially expressed in tumor tissues between GBMs and LGs, especially tRF-1-32-chrM.Lys-TTT, tiRNA-1-33-Gly-GCC-1, tiRNA-1-33-Gly-CCC-1, tRF-1-31-His-GTG-1, tiRNA-1-33-Gly-GCC-2-M3, and tiRNA-1-34-Lys-CTT-1-M2. These tsRNAs seemed to be associated with nucleotide excision repair, Hippo signaling, and cancer-related pathways. This may be the main reason for the differences in clinical characteristics between GBMs and LGs, which may provide a certain theoretical basis for further functional research and development of related nucleic acid drugs., The tsRNAs were differentially expressed in tumor tissues between GBMs and LGs, especially tRF-1-32-chrM.Lys-TTT, tiRNA-1-33-Gly-GCC-1, tiRNA-1-33-Gly-CCC-1, tRF-1-31-His-GTG-1, tiRNA-1-33-Gly-GCC-2-M3, and tiRNA-1-34-Lys-CTT-1-M2. These tsRNAs seemed to be associated with nucleotide excision repair, Hippo signaling, and cancer-related pathways. This may be the main reason for the differences in clinical characteristics between GBMs and LGs, which may provide a certain theoretical basis for further functional research and development of related nucleic acid drugs.