Stacks is a software system designed to identify and genotype loci in individuals using short-read sequence data, either de novo or by comparing to a reference genome. It is particularly useful for generating ultra-dense genetic linkage maps, examining population phylogeography, and aiding in reference genome assembly. The software uses a maximum likelihood statistical model to identify sequence polymorphisms and distinguish them from sequencing errors. Stacks can handle large numbers of markers and is implemented in C++ and Perl, with a web interface for data visualization and annotation. The effectiveness of Stacks is demonstrated through simulations and the construction of a high-density genetic map of zebrafish (Danio rerio) using RAD-tag data. The results show that Stacks can accurately recover loci and genotypes, even at high error rates and low sequencing depths.Stacks is a software system designed to identify and genotype loci in individuals using short-read sequence data, either de novo or by comparing to a reference genome. It is particularly useful for generating ultra-dense genetic linkage maps, examining population phylogeography, and aiding in reference genome assembly. The software uses a maximum likelihood statistical model to identify sequence polymorphisms and distinguish them from sequencing errors. Stacks can handle large numbers of markers and is implemented in C++ and Perl, with a web interface for data visualization and annotation. The effectiveness of Stacks is demonstrated through simulations and the construction of a high-density genetic map of zebrafish (Danio rerio) using RAD-tag data. The results show that Stacks can accurately recover loci and genotypes, even at high error rates and low sequencing depths.