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.