The use of phylogenetic methods to reconstruct the evolutionary history of molecular sequences is a vital part of biological research. These methods underpin studies on the evolution and epidemiology of plant and animal parasites and disease-causing organisms, and more generally they allow an objective analysis of the patterns and processes generating biological diversity. To carry out phylogenetic analysis properly, it is necessary to have a good understanding of the strengths and weaknesses of the key methods and approaches. This EMBO Practical Course has been designed to provide the theoretical and practical skills needed to carry out state-of-the-art phylogenetic analyses.