This book constitutes the refereed proceedings of the 16th International Workshop on Algorithms in Bioinformatics, WABI 2016, held in Aarhus, Denmark. The 25 full papers together with 2 invited talks presented were carefully reviewed and selected from 54 submissions. The selected papers cover a wide range of topics from networks, to phylogenetic studies, sequence and genome analysis, comparative genomics, and mass spectrometry data analysis. .