The subfamily Steninae, composed of the genera Dianous Leach 1819 and Stenus Latreille 1797, belongs to the family of staphylinid beetles (Staphylinidae). Some unique features characterize Stenus beetles, e.g., a distinct prey-capture apparatus (not found in the genus Dianous) and special pygidial gland secretion constituents such as the alkaloids stenusine (1), norstenusine (2), 3-(2-methyl-1-butenyl)pyridine (3), cicindeloine (4) as well as several terpenes like a-pinene (5), 1,8-cineole (6) and 6-methyl-5-heptene-2-one (7); (only 1, 2 and terpenes found in Dianous). As the secretion composition of Stenus beetles is species specific, it can be used for a chemotaxonomic approach to investigate relationships within the Steninae. Based on the alkaloid gland content, Steninae can be grouped into three clusters: the piperidine-, the pyridineand the epoxypiperideine group. To clarify the phylogenetic relationships within Stenus and between Stenus and Dianous, and to evaluate our chemotaxonomic approach, we analyzed a combined dataset of three gene sequences aligned of mitochondrial cytochrome c oxidase I (COI), 16S rRNA and Histone H3 of 17 Stenus and 4 Dianous species. Our investigations based on two algorithms such as Maximum Likelihood and Bayesian analyses support the chemotaxonomic approach. Furthermore, our results clearly support former analyses concerning the evolutionary origin of Dianous within Stenus, which suggests a secondary loss of the specialized prey-capture apparatus. Finally, phylogenetic aspects based on the morphology of Steninae are discussed.