Using mtDNA sequences we found that the Indo-Pacific teatfish fishery comprises at least three species, clarifying confusion on the taxonomic status of these commercially important holothurians. Traditional taxonomic characters, including the morphology of skeletal structures, could not be used to differentiate the species. Sequences of the COI gene (529 bp) distinguished three haplotype clusters, corresponding to distinct colour forms and, to some extent, previously described species. The white teatfish, Holothuria fuscogilva, comprises a range of colour morphs and has a wide distribution over the tropical Indo-Pacific region. The large sequence divergence indicates potential for the presence of several cryptic species in the white teatfish complex. In contrast to current taxonomy, we identified two species of black teatfish that appear to be allopatric: H. whitmaei is entirely black and has a Pacific distribution; whereas H. nobilis has white ventro-lateral dots and only occurs in the Indian Ocean. There is evidence for allopatric speciation between the black teatfish species, possibly driven by separation of the oceans and altered current patterns during the Pliocene, resulting in relatively young species with low intraspecific sequence divergence. These results provide insight into speciation in these tropical holothurians and are crucial for their conservation management.