Lancelets ("amphioxus") are the modern survivors of an ancient chordate lineage, the cephalochordates, with a fossil record dating back to the Cambrian. We describe the structure and gene content of the highly polymorphic genome of the Florida lancelet, in the context of the known genomes from the other chordate lineages, including the tunicate Ciona, human, and other vertebrates. This whole-genome comparison illuminates the murky relationship between the three principal chordate subphyla (tunicates, cephalochordates, and vertebrates), and provides a new perspective on the nature of the chordate common ancestor and key events in the emergence of vertebrates. The amphioxus sequence allows reconstruction of not only the gene complement of the ancestral chordate, but also a partial reconstruction of its genomic organization and a description of subsequent duplications and reorganizations in the vertebrate lineage.