DnaSP v5 is a software package for comprehensive analysis of DNA polymorphism data. It allows extensive DNA polymorphism analyses on large datasets and includes new features such as analysis on multiple data files, haplotype phasing, analysis of insertion/deletion (indel) polymorphism data, and visualization of sliding window results integrated with genome annotations in the UCSC browser. The software provides a user-friendly graphical interface (GUI) for data management and analysis, and results can be visualized graphically or in text reports. DnaSP supports multiple DNA sequence alignment file formats, including NEXUS and HapMap3 files with phased haplotypes. It enables exhaustive DNA polymorphism analyses, including those based on coalescent theory. Haplotype reconstruction in DnaSP uses statistical methods to infer haplotype phase from genotypic information. It applies various algorithms (PHASE, fastPHASE, and HAPAR) to determine haplotype phase, with PHASE assuming Hardy-Weinberg equilibrium and using a Bayesian method, fastPHASE being faster and suitable for larger datasets, and HAPAR using maximum parsimony to infer haplotype phase. DnaSP also analyzes deletion/insertion polymorphisms (DIPs) using an algorithm based on the 'simple indel coding' method. It estimates DIP summary statistics, such as average indel length, indel diversity, and Tajima's D, and exports recoded data in NEXUS format. DnaSP can process multiple data files in batch mode, estimating common DNA polymorphism and divergence summary statistics, as well as neutrality tests. It also allows visualization of sliding window results integrated with genome annotations in the UCSC browser, facilitating the interpretation of results by identifying relevant genome annotations. DnaSP v5 is developed in Microsoft Visual Basic v6.0, C, and C++, and runs on Microsoft Windows, Apple Macintosh, Linux, and Unix-based operating systems. It has been tested on all three platforms. The software is freely available to academic users. Funding was provided by the Spanish Dirección General de Investigación Científica y Técnica and the Catalonian Comissió Interdepartmental de Recerca i Innovació Tecnològica. No conflicts of interest were declared.DnaSP v5 is a software package for comprehensive analysis of DNA polymorphism data. It allows extensive DNA polymorphism analyses on large datasets and includes new features such as analysis on multiple data files, haplotype phasing, analysis of insertion/deletion (indel) polymorphism data, and visualization of sliding window results integrated with genome annotations in the UCSC browser. The software provides a user-friendly graphical interface (GUI) for data management and analysis, and results can be visualized graphically or in text reports. DnaSP supports multiple DNA sequence alignment file formats, including NEXUS and HapMap3 files with phased haplotypes. It enables exhaustive DNA polymorphism analyses, including those based on coalescent theory. Haplotype reconstruction in DnaSP uses statistical methods to infer haplotype phase from genotypic information. It applies various algorithms (PHASE, fastPHASE, and HAPAR) to determine haplotype phase, with PHASE assuming Hardy-Weinberg equilibrium and using a Bayesian method, fastPHASE being faster and suitable for larger datasets, and HAPAR using maximum parsimony to infer haplotype phase. DnaSP also analyzes deletion/insertion polymorphisms (DIPs) using an algorithm based on the 'simple indel coding' method. It estimates DIP summary statistics, such as average indel length, indel diversity, and Tajima's D, and exports recoded data in NEXUS format. DnaSP can process multiple data files in batch mode, estimating common DNA polymorphism and divergence summary statistics, as well as neutrality tests. It also allows visualization of sliding window results integrated with genome annotations in the UCSC browser, facilitating the interpretation of results by identifying relevant genome annotations. DnaSP v5 is developed in Microsoft Visual Basic v6.0, C, and C++, and runs on Microsoft Windows, Apple Macintosh, Linux, and Unix-based operating systems. It has been tested on all three platforms. The software is freely available to academic users. Funding was provided by the Spanish Dirección General de Investigación Científica y Técnica and the Catalonian Comissió Interdepartmental de Recerca i Innovació Tecnològica. No conflicts of interest were declared.