This book constitutes the proceedings of the 20th Annual Conference on Research in Computational Molecular Biology, RECOMB 2016, held in Santa Monica, CA, USA, in April 2016. The 15 regular papers presented in this volume were carefully reviewed and selected from 172 submissions. 20 short abstracts are included in the back matter of the volume. They report on original research in all areas of computational molecular biology and bioinformatics.