This book constitutes the refereed proceedings of the 4th Brazilian Symposium on Bioinformatics, BSB 2009, held in Porto Alegre, Brazil, in July 2009. The 12 revised full papers and 6 extended abstracts were carefully reviewed and selected from 55 submissions. The papers are organized in topical sections on algorithmic approaches for molecular biology problems; micro-array analysis; machine learning methods for classification; and in silico simulation.