We present epiGBS2, a laboratory protocol based on epiGBS (Gurp et al., 2016) with a revised and user-friendly bioinformatics pipeline for a wide range of species with or without reference genome. Performance of several critical steps in epiGBS2 was evaluated against baseline data sets from Arabidopsis thaliana and Great tit (Parus major), which confirmed overall good performance of epiGBS2. We provide here the raw bisulfite sequencing data of the epiGBS2 run for Arabidopsis thaliana. A detailed description of the laboratory protocol and an extensive manual of the bioinformatics pipeline are publicly accessible on github (https://github.com/nioo-knaw/epiGBS2) and zenodo (https://doi.org/10.5281/zenodo.4764652). Demultiplexed data were deposited on NCBI under the BioProject ID PRJNA764918