A novel mitochondrial tRNA gene arrangement is described for two species of sea cucumber. The mitochondrial tRNA gene cluster common to sea stars, sea urchins, and the sea cucumber Parastichopus californicus has been significantly modified in the genus Cucumaria as a result of dispersal of the tRNA genes into two separate areas of the genome. The tRNA genes in the novel clusters are interspersed with short unassigned sequences (UASs). Alignment of the two separated novel clusters indicates that the rearrangement was most likely the result of a tandem duplication of approximately 7 kb, encompassing the putative control region, the tRNA cluster, NADH dehydrogenase subunits 1 and 2, the large ribosomal RNA (lrRNA), cytochrome oxidase subunit I, and tRNAArg. Subsequently, deletion of the duplicated lrRNA and protein-coding genes occurred. In addition, the degeneration of one of each of the duplicated tRNA gene pairs has resulted in the interspersed UAS segments observed in each cluster. In contrast, the second copy of the putative control region has been maintained with a very high degree of sequence conservation, suggesting either some functional constraint or concerted evolution for the duplicated element. Analysis of gene organization in other sea cucumber species may provide (1) important insights into the mechanism of mitochondrial gene rearrangements and (2) an informative character set for deep-level phylogenetic analysis of this echinoderm class.