The family Cymatosiraceae, composed of two subfamilies (Cymatosiroideae and Extubocelluloideae), are elongate, non-pennate diatoms and are commonly reported in marine planktonic and benthic assemblages all over the world. The combination of the gross bilateral symmetry of the frustule and radial symmetry of the valve poration, distinct pore fields at the valve apices and DNA sequence data place this family in the controversial diatom class Mediophyceae, sister to the pennate diatoms. The relationships of the Cymatosiraceae within the Mediophyceae, and the monophyly of the two cymatosiroid subfamilies, have become less stable with the addition of new DNA sequence data. In this paper, we examined 24 cymatosiraceaen strains, including the new taxa Lambertocellus africana (Dąbek & Witkowski) Dąbek, Witkowski & Ashworth comb. nov., Leyanella probus Ashworth, Dąbek & Witkowski sp. nov., and Leyanella pauciporis Ashworth, Dąbek & Park sp. nov. to create the most complete molecular phylogeny of the Cymatosiraceae to date, using a three-gene (SSU, rbcL and psbC) data set. The results of the phylogenetic analysis supported the monophyly of the Cymatosiraceae, but not the monophyly of the subfamilies Cymatosiroideae and Extubocelluloideae. The phylogenetic analysis also suggested a close relationship of the Cymatosiraceae to the family Eupodiscaceae. The phylogenetic results have lead us to re-evaluate the taxonomy of L. africana, which is sister to Cymatosira lorenziana rather than Minutocellus as suggested in the original description.