This chapter introduces the problem of ancestral sequence reconstruction: given a set of extant orthologous DNA genomic sequences (or even whole-genomes), together with a phylogenetic tree relating these sequences, predict the DNA sequence of all ancestral species in the tree. Blanchette et al. (1) have shown that for certain sets of species (in particular, for eutherian mammals), very accurate reconstruction can be obtained. We explain the main steps involved in this process, including multiple sequence alignment, insertion and deletion inference, substitution inference, and gene arrangement inference. We also describe a simulation-based procedure to assess the accuracy of the reconstructed sequences. The whole reconstruction process is illustrated using a set of mammalian sequences from the CFTR region.