Diatoms are a highly diverse and widespread group of unicellular algae. They are of global importance for the carbon and nitrogen cycles of marine and freshwater environments. Due to their high diversity, ubiquitous distribution and relatively good identifiability of the different species, diatoms are used as bioindicators, e.g. for the purpose of water quality assessments, tracking of environmental changes or paleoecological reconstruction of water bodies. Traditionally, species identification is done morphologically with the help of microscopes. DNA barcoding is an alternative or complementary methodology that identifies species using short, standardized DNA fragments. Metabarcoding, which is still under development for diatoms, uses this method to determine the species composition of environmental samples. To enable effective DNA barcoding of diatoms, some remaining challenges need to be addressed. Among other things, a correct taxonomy, a sufficiently complete reference database, which links DNA and morphology, and effective DNA barcoding markers are necessary. The aim of this work, using the example of thalassiosiroid diatoms, is to contribute to overcoming the mentioned problems. With the help of an extensive collection of cultures, several contributions to the taxonomy of the genera Cyclostephanos, Discostella, Stephanodiscus and Pantocsekiella could be made. Among other things, the knowledge of existing species was improved, new species were described, the taxonomic implications of the colony formation of some species were examined, and a high degree of cryptic diversity in the genus Discostella was discovered. In addition, the discriminatory power of four established DNA barcoding markers was tested on the basis of 277 cultures belonging to 55 species of thalassiosiroid diatoms. Furthermore, four new markers were developed and successfully tested. These new markers show a high discriminatory power between the different species. With more than 1250 created sequences, the results of this work represent a contribution to the completion of the DNA barcoding reference databases.