DNA Subway is a collaborative workspace to generate DNA sequence annotations, conduct phylogenetic analyses, and analyze Next-Generation Sequencing data. DNA Subway is an appealing and intuitive interface that uses the metaphor of a network of subway lines to guide users through the maze of analysis steps necessary to annotate and compare DNA sequences. Developed with an eye to undergraduate education; DNA Subway can make high-level DNA analysis available to faculty and students by simplifying annotation and comparative genomics workflows.