These proceedings contain papers from the 2009 Workshop on Algorithms in Bioinformatics (WABI), held at the University of Pennsylvania in Philadelphia, Pennsylvania during September 12–13, 2009. WABI 2009 was the ninth annual conference in this series, which focuses on novel algorithms that address imp- tant problems ingenomics, molecular biology, and evolution. The conference- phasizes research that describes computationally efficient algorithms and data structures that have been implemented and tested in simulations and on real data. WABI is sponsored by the European Association for Theoretical C- puter Science (EATCS) and the International Society for Computational Bi- ogy (ISCB). WABI 2009 was supported by the Penn Genome Frontiers Institute and the Penn Center for Bioinformatics at the University of Pennsylvania. For the 2009 conference, 90 full papers were submitted for review by the Program Committee, and from this strong field of submissions, 34 papers were chosen for presentation at the conference and publication in the proceedings. The final programcovered a wide range of topics including gene interaction n- works, molecular phylogeny, RNA and protein structure, and genome evolution.