DnaSP is a software package for analyzing DNA polymorphism data. The latest version includes new modules and features that allow handling large datasets (up to 5 Mb per sequence), conducting coalescent-based tests via Monte Carlo simulations, extensive analysis of genetic differentiation and gene flow among populations, analysis of codon usage bias, and generating graphical outputs. The software is available for academic use at http://www.ub.es/dnasp and can be run on Windows, Macintosh, Linux, and Unix platforms. DnaSP version 4 is written in Microsoft Visual Basic 6.0 and runs on Windows-compatible processors. It supports five nucleotide sequence file formats: FASTA, MEGA, NBRF/PIR, NEXUS, and PHYLIP. It allows analysis of polymorphism, divergence, genetic differentiation, gene flow, gene conversion, linkage disequilibrium, recombination, and codon usage, as well as neutrality tests. It can analyze subsets of sites or sequences and uses predefined genetic codes and codon usage tables for coding region analysis. DnaSP has enhanced coalescent-based analyses, allowing most neutrality tests (with and without outgroup) and linkage disequilibrium statistics, including Tajima's, Fu's, and Fu and Li's tests, Depaulis and Veuille's haplotype-based tests, B- and Q-tests, H-test, and Z-based statistics. It also computes population growth tests, including the R2 test. Monte Carlo simulations generate empirical distributions for test statistics and can be conducted for different recombination rates. The Gene Flow module allows analysis of gene flow and genetic differentiation among populations, using statistics based on haplotype numbers and nucleotide changes. It estimates FST, GST, and NST parameters and calculates migration rates. It also provides two methods to test for genetic differentiation: a chi-square test and a Monte Carlo permutation test. DnaSP analyzes the impact of natural selection and mutational processes on codon usage bias. It identifies preferred and unpreferred synonymous changes and estimates the numbers of preferred and unpreferred changes within and between species. It provides predefined codon usage tables and allows users to define their own. This work was supported by grants from the Spanish government.DnaSP is a software package for analyzing DNA polymorphism data. The latest version includes new modules and features that allow handling large datasets (up to 5 Mb per sequence), conducting coalescent-based tests via Monte Carlo simulations, extensive analysis of genetic differentiation and gene flow among populations, analysis of codon usage bias, and generating graphical outputs. The software is available for academic use at http://www.ub.es/dnasp and can be run on Windows, Macintosh, Linux, and Unix platforms. DnaSP version 4 is written in Microsoft Visual Basic 6.0 and runs on Windows-compatible processors. It supports five nucleotide sequence file formats: FASTA, MEGA, NBRF/PIR, NEXUS, and PHYLIP. It allows analysis of polymorphism, divergence, genetic differentiation, gene flow, gene conversion, linkage disequilibrium, recombination, and codon usage, as well as neutrality tests. It can analyze subsets of sites or sequences and uses predefined genetic codes and codon usage tables for coding region analysis. DnaSP has enhanced coalescent-based analyses, allowing most neutrality tests (with and without outgroup) and linkage disequilibrium statistics, including Tajima's, Fu's, and Fu and Li's tests, Depaulis and Veuille's haplotype-based tests, B- and Q-tests, H-test, and Z-based statistics. It also computes population growth tests, including the R2 test. Monte Carlo simulations generate empirical distributions for test statistics and can be conducted for different recombination rates. The Gene Flow module allows analysis of gene flow and genetic differentiation among populations, using statistics based on haplotype numbers and nucleotide changes. It estimates FST, GST, and NST parameters and calculates migration rates. It also provides two methods to test for genetic differentiation: a chi-square test and a Monte Carlo permutation test. DnaSP analyzes the impact of natural selection and mutational processes on codon usage bias. It identifies preferred and unpreferred synonymous changes and estimates the numbers of preferred and unpreferred changes within and between species. It provides predefined codon usage tables and allows users to define their own. This work was supported by grants from the Spanish government.