In this lesson, students learn how to use bioinformatics tools to analyze DNA sequence data and draw conclusions about evolutionary relationships. Students collaborate with their group members by pooling their DNA sequences from Lesson Two: DNA Barcoding and the Barcode of Life Database (BOLD) to perform and analyze multiple sequence alignments using the computer programs ClustalW2 and JalView. After comparing relatedness among and between the species within their group, students use their sequence alignment to generate a phylogenetic tree, which is a graphical representation of inferred evolutionary relationships. This tree is used to draw conclusions about their research question and hypothesis. In Lesson Three, students learn how microbiologists might use bioinformatics tools in their career.