DnaSP 6 is a new version of the DNA Sequence Polymorphism (DnaSP) software, designed for comprehensive population genetic analysis of multiple sequence alignments. This major upgrade incorporates novel functionalities to analyze large data sets, especially those generated by high-throughput sequencing technologies. Key features include modules for reading and analyzing data from genomic partitioning methods like RADseq or hybrid enrichment approaches, faster methods scalable for high-throughput sequencing data, and summary statistics for multi-locus population genetics data. DnaSP 6 also includes new modules for single- and multi-locus coalescent simulations under various demographic scenarios. DnaSP 6 can efficiently process and analyze massive data generated by programs such as PyRAD and STACKS, including *.alleles, *.loci, and *.fa format files. It can also handle data from other partitioning approaches, including low-coverage whole-genome sequencing projects and VCF files. The software can perform comprehensive population genetic analyses using multi-MSA files, estimating a large set of statistics that summarize DNA sequence variation within and between populations, including linkage disequilibrium and gene flow. It also estimates individual heterozygosity from genotype data and incorporates new neutrality tests, including Zengs E and Achaz's Y* and Y. DnaSP 6 is capable of processing the Arlequin format, which stores haplotype frequency information. The coalescent theory is extended to analyze DNA samples under the coalescent, incorporating algorithms from Hudson and Ramos-Onsins and Mitchell-Olds. The new coalescent modules automatically capture summary statistics from observed data, using single-locus mode or batch routines for multi-locus analyses. The software can evaluate the likelihood of summary statistics under various demographic scenarios. DnaSP 6 was migrated from Visual Basic 6 to VB.NET, supporting multi-threading, optimizing RAM usage, enabling 64-bit variables, and facilitating interoperability with the Internet. It was benchmarked using diverse data sets and configurations, showing efficient management of large data files. The software can perform a complete DNA Polymorphism analysis in a few seconds, making it suitable for analyzing representative data files from various genome partitioning methods.DnaSP 6 is a new version of the DNA Sequence Polymorphism (DnaSP) software, designed for comprehensive population genetic analysis of multiple sequence alignments. This major upgrade incorporates novel functionalities to analyze large data sets, especially those generated by high-throughput sequencing technologies. Key features include modules for reading and analyzing data from genomic partitioning methods like RADseq or hybrid enrichment approaches, faster methods scalable for high-throughput sequencing data, and summary statistics for multi-locus population genetics data. DnaSP 6 also includes new modules for single- and multi-locus coalescent simulations under various demographic scenarios. DnaSP 6 can efficiently process and analyze massive data generated by programs such as PyRAD and STACKS, including *.alleles, *.loci, and *.fa format files. It can also handle data from other partitioning approaches, including low-coverage whole-genome sequencing projects and VCF files. The software can perform comprehensive population genetic analyses using multi-MSA files, estimating a large set of statistics that summarize DNA sequence variation within and between populations, including linkage disequilibrium and gene flow. It also estimates individual heterozygosity from genotype data and incorporates new neutrality tests, including Zengs E and Achaz's Y* and Y. DnaSP 6 is capable of processing the Arlequin format, which stores haplotype frequency information. The coalescent theory is extended to analyze DNA samples under the coalescent, incorporating algorithms from Hudson and Ramos-Onsins and Mitchell-Olds. The new coalescent modules automatically capture summary statistics from observed data, using single-locus mode or batch routines for multi-locus analyses. The software can evaluate the likelihood of summary statistics under various demographic scenarios. DnaSP 6 was migrated from Visual Basic 6 to VB.NET, supporting multi-threading, optimizing RAM usage, enabling 64-bit variables, and facilitating interoperability with the Internet. It was benchmarked using diverse data sets and configurations, showing efficient management of large data files. The software can perform a complete DNA Polymorphism analysis in a few seconds, making it suitable for analyzing representative data files from various genome partitioning methods.