SUMMARY: We present Nubeam-dedup, a fast and RAM-efficient tool to de-duplicate sequencing reads without reference genome. Nubeam-dedup represents nucleotides by matrices, transforms reads into products of matrices, and based on which assigns a unique number to a read. Thus, duplicate reads can be efficiently removed by using a collisionless hash function. Compared with other state-of-the-art reference-free tools, Nubeam-dedup uses 50-70% of CPU time and 10-15% of RAM. AVAILABILITY AND IMPLEMENTATION: Source code in C++ and manual are available at https://github.com/daihang16/nubeamdedup and https://haplotype.org. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.