DnaSP is a software package for the analysis of DNA polymorphism data. Present version introduces several new modules and features which, among other options allow: (1) handling big data sets (approximately 5 Mb per sequence); (2) conducting a large number of coalescent-based tests by Monte Carlo computer simulations; (3) extensive analyses of the genetic differentiation and gene flow among populations; (4) analysing the evolutionary pattern of preferred and unpreferred codons; (5) generating graphical outputs for an easy visualization of results.