Based on morphological, chemical and phylogenetic analyses, three species of Ochrolechia collected from Guizhou, China, are described as new to science and named Ochrolechia guizhouensis, O. kuankuoshuiensis and O. leigongshanensis. Phylogenetic trees, based on ITS and mtSSU loci, were constructed using Maximum Likelihood analysis (ML) and Bayesian Inference (BI) methods. Ochrolechia guizhouensis and O. akagiensis are positioned close to each other in the phylogenetic tree. Ochrolechia guizhouensis is morphologically characterised by its apothecia which feature rugose to rosulate, epruinose discs resembling a floral structure. Chemically, the apothecia contain gyrophoric acid, lecanoric acid and lichesterinic acid, while the thallus contains only gyrophoric acid and lecanoric acid. Phylogenetic analysis reveals that Ochrolechia kuankuoshuiensis is closely related to O. parellula and is distinguished by its light yellow, epruinose, deeply rugose apothecial discs, the production of gyrophoric acid and lecanoric acid and the largest ascospores known in the genus. Ochrolechia leigongshanensis forms an isolated phylogenetic branch which is characterised by a soraliate thallus and apothecia with a ring of smooth, salmon-pink tissue on the inner margin. The discs are epruinose to lightly pruinose and plane. The thallus contains gyrophoric acid, lecanoric acid and a trace of atranorin, while the apothecia contain only gyrophoric acid and lecanoric acid. Additionally, O. subrosella and O. longispora were successfully sequenced for the first time and represent the first records from Guizhou Province. We found one specimen, which phylogenetically groups with O. trochophora, but is characterised by the presence of soralia, indicating that O. trochophora might be morphologically more variable than previously thought. Colour photographs are provided for all the above-mentioned species.