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The lichenized fungal genus <i>Astrothelium</i> is an important element of crustose lichen communities in tropical to subtropical forests. Morphological and molecular phylogenetic approaches to investigate species diversity of <i>Astrothelium</i> (<i>Trypetheliaceae</i>) from Southern China were carried out in this study. Bayesian and maximum-likelihood (ML) analyses were generated based on the combined data set of internal transcribed spacer (ITS), partial regions of the nuclear ribosomal large subunit (LSU), and the largest subunit of RNA polymerase II gene sequences (RPB1). The morphological comparison with the known <i>Astrothelium</i> taxa and molecular phylogeny support five new species: <i>Astrothelium jiangxiense</i> sp. nov., <i>A. luminothallinum</i> sp. nov., <i>A. pseudocrassum</i> sp. nov., <i>A. subeustominspersum</i> sp. nov., and <i>A</i><i>. subrufescens</i> sp. nov. All these species are described and illustrated in detail.