Chaetoceros Ehrenberg is one of the most diverse genera of planktonic diatoms. The species in section Chaetoceros are characterized by cells and setae having numerous chloroplasts and being widely distributed. However, the delimitations of some species are problematic because of limited morphological information in the classical descriptions. Monoclonal strains of the section Chaetoceros were established, morphological features were studied using light and electron microscopy, and the hypervariable D1–D3 region of the nuclear ribosomal large subunit gene was sequenced to address phylogenetic relationships. Fifteen species belonging to the section Chaetoceros were recorded, including two new species, C. hainanensis sp. nov. and C. tridiscus sp. nov. Chaetoceros hainanensis was characterized by straight chains, narrowly lanceolate to hexagonal apertures, sibling setae diverging in nearly right angles, stipule-shaped spines on terminal setae and arrowhead-shaped spines on intercalary setae. C. tridiscus had short straight chains, narrowly lanceolate apertures, arrowhead-shaped spines and circular poroids arranged in a grid pattern on terminal and intercalary setae. The phylogenetic analyses revealed six groups formed by 19 species within the section Chaetoceros, which was found to be monophyletic. The subdivision of the section is still not well understood. The morphological characters within each group varied considerably and molecular information on more species are needed to enrich the phylogenetic profiling.