Here we present detailed morphological and molecular comparison of the Tonicia species and ecotypes along Southeastern Pacific coast. Several specimens were examined by scanning electron microscopy and based on plates morphology we propose a new taxonomic key for identification of species and ecotypes of Tonicia. A total of 566 specimens were measured to explore morphological differences between ecotypes and ecoregions by means of multivariate analyses. Genetic distances and a maximum likelihood (ML) phylogeny were estimated using Cytochrome Oxidase I (cox1) to compare species and ecotypes. Morphometry and multivariate analyses revealed morphological differences between ecotypes in each species. However, cox1 genetic distances were low between ecotypes in both species (T. calbucensis and T. chilensis) and the ML phylogeny revealed an absence of monophyletic relationships between ecotypes in each species clades. The new key will be useful to identify Tonicia species by morphology without molecular analysis.