UNLABELLED: An increasingly common method for studying evolution is the collection of time-resolved short-read sequence data. Such datasets allow for the direct observation of rapid evolutionary processes, as might occur in natural microbial populations and in evolutionary experiments. In many circumstances, evolutionary pressure acting upon single variants can cause genomic changes at multiple nearby loci. SAMFIRE is an open-access software package for processing and analyzing sequence reads from time-resolved data, calling important single- and multi-locus variants over time, identifying alleles potentially affected by selection, calculating linkage disequilibrium statistics, performing haplotype reconstruction and exploiting time-resolved information to estimate the extent of uncertainty in reported genomic data. AVAILABILITY AND IMPLEMENTATION: C ++ code may be found at https://github.com/cjri/samfire/ CONTACT: chris.illingworth@gen.cam.ac.uk SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.