ABSTRACT: Small non-coding RNAs (sncRNAs) are generated from different genomic loci and have important roles in biological processes, such as cell proliferation and regulation of gene expression. Next-generation sequencing (NGS) has provided an unprecedented opportunity to discover and quantify diverse kinds of sncRNAs, such as tRFs (tRNA-derived small RNA fragments), phasiRNAs (phased, secondary, small interfering RNAs), piRNAs and plant specific 24-nt short interfering RNAs (siRNAs). However, currently available web-based tools do not provide approaches to comprehensively analyze all these diverse sncRNAs. A novel web server, sRNAtools (https://bioinformatics.sc.cn/sRNAtools), is presented that can be used in conjunction with high-throughput sequencing to discover, profile and functionally annotate sncRNAs, including miRNAs, isomiRs, piRNAs, tRFs, tRNAs, snRNAs, snoRNAs, rRNAs, phasiRNAs, and plant specific 24-nt siRNAs for 21 species. In this study, sRNAtools has been used in sncRNA profiling in poplar wood tissues, which identified many wood related sncRNAs expression patterns.We believe that sRNAtools will greatly benefit the scientific community as an integrated tool to study sncRNAs.