The mitochondrial genomes (mtDNA) of Tunicata, basal chordates and sister group to vertebrates, seem to follow different evolutionary dynamics to those of other chordates, characterized by many gene rearrangements and accelerated evolutionary rates. In order to investigate the peculiarities of mtDNA evolution in tunicates, we amplified (by long PCR) and completely sequenced the mtDNA of five ascidians: two Stolidobranchia species, Microcosmus sulcatus (Pyuridae) and Styela plicata (Styelidae); two cogeneric Phlebobranchia species, Phallusia mammillata and Phallusia fumigata (Ascidiidae); and a Ciona intestinalis individual isolated in Plymouth, UK. The analyses confirmed previous observations of a high rate of gene rearrangement in these genomes. Furthermore, base compositional variability, shortness of rRNA genes, and absence of a main non-coding region were confirmed as common features of ascidian mitochondrial genomes and indicate that the evolutionary dynamics of ascidian mtDNA markedly diverge from those of vertebrates. Finally, we used a mitogenomic approach to unambiguously demonstrate the existence of two cryptic species in Ciona intestinalis, currently described as a single species. A comprehensive comparative analysis between the mtDNAs of two types of C. intestinalis revealed strong differences in gene order, size/number of non-coding regions, base composition, codon usage, and evolutionary rate of protein-coding genes. All such features are incompatible with intra-specific variability, and concur with the existence of two cryptic species in this model organism. Furthermore, our mitogenomic approach allowed the development of fast diagnostic tests for the discrimination of the cryptic species without recourse to morphological analyses, demonstrating that mtDNA represents an accessible and powerful tool to be used in routine taxonomic analyses.