A comprehensive set of computational tools for evolutionary analysis of whole-genome alignments consisting of multiple individuals, from multiple populations or species. POPBAM works directly from BAM-formatted assembly files, calls variant sites, and calculates a variety of commonly used evolutionary sequence statistics. POPBAM is designed primarily to perform analyses in sliding windows across chromosomes or scaffolds. POPBAM accurately measures nucleotide diversity, population divergence, linkage disequilibrium, and the frequency spectrum of mutations from two or more populations. POPBAM can also produce phylogenetic trees of all samples in a BAM file.