Stacks is a computational tool for population genomics that efficiently processes genotype-by-sequencing (GBS) data. It enables the analysis of tens of thousands of genetic markers, providing core population genomic summary statistics and SNP-by-SNP statistical tests. Stacks can analyze data across a reference genome using a smoothed sliding window and offers several output formats for downstream analysis packages. The software is robust, efficient, and flexible, capable of handling data from thousands of individuals and incorporating a MySQL database and web interface for data visualization and management. Stacks was initially developed for genetic mapping crosses but has been expanded for ecological and evolutionary genomic analyses. It now supports de novo stack formation, reference-guided stack formation, and SNP calling using a bounded-error model. Stacks also provides population genetic statistics such as π, FIS, and FST, and allows kernel smoothing of reference-aligned statistics. It can perform genome-wide tests of statistical significance using bootstrap resampling and export data in formats compatible with other evolutionary genomic programs. Stacks is primarily written in C++ and includes Perl scripts for common tasks, and is parallelized for efficient processing. It can handle any restriction digest-based data and produces core population genomic summary statistics and SNP-by-SNP statistical tests. Stacks is a key resource for researchers to efficiently perform ecological and evolutionary genomic studies, particularly in organisms with minimal genomic resources. The software is continuously being expanded and improved in line with developments in population genomics.Stacks is a computational tool for population genomics that efficiently processes genotype-by-sequencing (GBS) data. It enables the analysis of tens of thousands of genetic markers, providing core population genomic summary statistics and SNP-by-SNP statistical tests. Stacks can analyze data across a reference genome using a smoothed sliding window and offers several output formats for downstream analysis packages. The software is robust, efficient, and flexible, capable of handling data from thousands of individuals and incorporating a MySQL database and web interface for data visualization and management. Stacks was initially developed for genetic mapping crosses but has been expanded for ecological and evolutionary genomic analyses. It now supports de novo stack formation, reference-guided stack formation, and SNP calling using a bounded-error model. Stacks also provides population genetic statistics such as π, FIS, and FST, and allows kernel smoothing of reference-aligned statistics. It can perform genome-wide tests of statistical significance using bootstrap resampling and export data in formats compatible with other evolutionary genomic programs. Stacks is primarily written in C++ and includes Perl scripts for common tasks, and is parallelized for efficient processing. It can handle any restriction digest-based data and produces core population genomic summary statistics and SNP-by-SNP statistical tests. Stacks is a key resource for researchers to efficiently perform ecological and evolutionary genomic studies, particularly in organisms with minimal genomic resources. The software is continuously being expanded and improved in line with developments in population genomics.