This unit provides a general description of reconstructing evolutionary trees using PAUP* 4.0. The protocol takes users through an example analysis of mitochondrial DNA sequence data using the parsimony and the likelihood criteria to infer optimal trees. The protocol also discusses searching options available in PAUP* and demonstrates how to import non-NEXUS formats. Finally, a general discussion is given regarding the pros and cons of the "model-free" and "model-based" methods used throughout the protocol.