A new species of the synallactid sea cucumber genus Pseudostichopus is described, P. aemaulatus sp. nov., based on genetic (DNA sequences of the mitochondrial gene Cytochrome Oxidase I [COI] gene) and morphological characters. A comparative molecular study with two other species of the same genus (P villosus and P. mollis) and from a different: Family (Isostichopus fuscus) was carried out in order to clarify its taxonomic identity. The nucleotide distance between P. aemulatus sp. nov. and P. villosus and P. mollis is sufficient to support distinct species status. The estimated difference in the number of amino acids, coded for by a partially sequenced COI gene, within the species of the family Synallactidae ranged from 4 to 18. The phylogenetic analysis clearly supports separate species status of these sympatric morphotypes, as indicated by the morphological analysis.