We present version 6 of the DnaSP (DNA Sequence Polymorphism) software, a new version of the popular tool for performing exhaustive population genetic analyses on multiple sequence alignments. This major upgrade incorporates novel functionalities to analyse large datasets, such as those generated by high-throughput sequencing (HTS) technologies. Among other features, DnaSP 6 implements: i) modules for reading and analysing data from genomic partitioning methods, such as RADseq or hybrid enrichment approaches, ii) faster methods scalable for HTS data, and iii) summary statistics for the analysis of multi-locus population genetics data. Furthermore, DnaSP 6 includes novel modules to perform single- and multi-locus coalescent simulations under a wide range of demographic scenarios. The DnaSP 6 program, with extensive documentation, is freely available at http://www.ub.edu/dnasp.