AbstractFive new species and one new record of Astrothelium (Trypetheliaceae) are reported from Thailand. Phylogenetic analysis of four DNA loci (ITS, nuLSU, mtSSU rDNA and RPB1) demonstrated the placement of the new species within Astrothelium, as well as their distinction from similar or related taxa. The new species are: A. flavocoronatum, with a yellow pigment surrounding the ostiole, and 3-septate ascospores 22–28×8·0–9·5 µm; A. macrostiolatum, with large whitish ostiolar area, a hamathecium inspersed with small oil droplets, and 9–11-septate ascospores 80–100×17–19 µm; A. neglectum, with lichexanthone on the thallus and pseudostromata, a non-inspersed hamathecium and 3–5-septate ascospores 21–25×7·5–9·5 µm; A. neovariolosum, with inspersed hamathecium and 3-septate ascospores 17–23×6–7 µm; and A. siamense, with inspersed hamathecium and 4–7-septate ascospores 30–50×10·5–12·0 µm. Astrothelium aenascens is reported from Thailand for the first time.