This paper describes the first study of the diatom assemblages in caves in the Czech Republic. The study focused on subaeric habitats: rock faces within caves, walls at cave entrances and “lampflora” assemblages, in three cave systems, Mladeč, Javoříčko and Zbrašov, all in central Moravia. The morphological and cytological variability, ecology and life strategies of diatoms were studied in fresh samples, in Naphrax preparations, in cultures grown on agar plates and in monoclonal cultures. A total of 22 diatom species was identified, mostly aerophytic species and tolerant of low light intensities. Luticola and Diadesmis species complexes are discussed. Taxa D. gallica and L. paramutica var. binodis are new for the Czech Republic. Sexual reproduction was observed in L. mutica and Orthoseira roeseana.