Chaetoceros is one of the most species-rich phytoplankton genera, and its known diversity can be expected to increase significantly when additional material from tropical and other less studied areas has been examined by a combination of molecular and morphological methods. We have succeeded in culturing and examining a pair of very characteristic Chaetoceros species, Chaetoceros compressus and Chaetoceros contortus, from a number of geographically separate localities. The pair is easily recognizable as such because of the heavy setae characteristic of the section Compressa but morphological similarities between the two species have caused considerable taxonomic confusion since the second species, C. contortus, was described nearly one hundred years ago. Critical studies exploring species delineation are therefore needed to determine whether C. compressus and C. contortus should be considered separate species. A combination of phylogenetic analyses based on large subunit rDNA and detailed morphological studies using light microscopy, scanning electron microscopy and transmission electron microscopy on strains of both species showed them to make up separate species and found that the variety C. compressus var. hirtisetus should be raised to species level as C. hirtisetus comb. & stat. nov. We describe two varieties of C. contortus: the temperate cf. var. contortus and the tropical var. ornatus var. nov. Characters for species delineation comprise a combination of light microscopically visible features, such as valve size and shape and number of chloroplasts. Furthermore, resting spore morphology and the number of labiate processes on the terminal valve seen in electron microscopy (EM) can be used to separate the two species, as well as molecular characters.