Thalassiosira and related taxa i.e. the taxa within Thalassiosirales are among the most abundant and widespread diatoms. The vast majority have one marginal rimoportula, but very few always have two. The knowledge on Thalassiosiroid taxa in Chinese waters is restricted, and to explore the diversity, monoclonal strains were established from southern Chinese coastal waters. Morphological data were based on light and scanning electron microscopy. The D1‒D3 region of the ribosomal large subunit (LSU) and the small subunit (SSU) encoding genes were amplified to address phylogenetic relationships. Based on a combination of morphological characters and molecular analyses, two new species with two marginal rimoportulae were described, Planktoniella pulchra sp. nov. and Thalassiosira tridens sp. nov. Planktoniella pulchra is furthermore characterized by one regular ring of marginal fultoportulae between the valve face and the mantle, as well as dense ribs on the mantle. A narrow circular extension of mucilaginous material surrounds the girdle. In phylogenetic analyses, it clustered within the Planktoniella group. Thalassiosira tridens is unique in having a pronounced external tube of the central fultoportula, as well as long external tubes of the two marginal rimoportulae. Two irregular rings formed by the marginal fultoportulae are present on the mantle, and their external extensions are either open tubes or closed spines. In the SSU and LSU phylogenetic analyses, both taxa constituted their own monophyletic lineage and were well differentiated from allied species. Thalassiosiroid taxa with two marginal rimoportulae were phylogenetically closely related, but did not make up a monophyletic lineage.