A new species, Astrothelium subsiamense from China, is described based on morphological, chemical, and molecular analyses. The new lichen is most similar to A. siamense but differs in its 3-septate and smaller ascospores. Its relationship with other Astrothelium spp. is presented, based on molecular phylogeny, and a key to the Astrothelium species recorded in China is also provided. Key words—lichenized fungi, taxonomy, South China, Trypetheliaceae, Trypetheliales.