Processing of mature transfer RNAs (tRNAs) produces complex populations of tRNA-derived fragments (tRFs). Emerging evidence shows that tRFs have important functions in bacteria, ani-mals, and plants. Here, we review recent ad-vances in understanding plant tRFs, focusing on their biological and cellular functions, such as regulating stress responses, mediating plant–pathogen interactions, and modulating post-transcriptional gene silencing and translation. We also review sequencing strategies and bio-informatics resources for studying tRFs in plants. Finally, we discuss future directions for plant tRF research, which will expand our knowledge of plant non-coding RNAs.