ABSTRACT We have sequenced a portion of the mitochondrial 16S rRNA gene from 29 species of Anthozoa, representing six orders of the subclasses Ceriantipatharia, Hexacorallia, and Octocorallia, with the focus on deep-seamount corals (> 500-m depth). We have detected significant length variation in the gene, with homologous gene fragments ranging from 545 bp in a shallow-water scleractinian coral to 911 bp in a deep-sea antipatharian black coral. The aligned sequences were divided into five regions: three high-identity sequence blocks (HSBs) and two highly variable blocks of insertions/deletions (INDELs). Most of the length variation among species occurred as varying numbers of nucleotides in the two INDELs. Little or no intraspecific sequence variation was detected over spatial scales of up to approximately 150 km. Interspecific sequence variation was lowest among the octocorals and greatest among the ceriantipatharians. Our data indicate that the orders Ceriantharia and Antipatharia are highly divergent, and a phylogenetic reconstruction provides support for the two-subclass system of the class Anthozoa (Hexacorallia and Octocorallia).