The Pearl River Basin, China’s second-largest freshwater basin, hosts a significant diversity of species and a highly endemic freshwater mussel fauna. In this study, a new species from the Liuxi River in Guangzhou, Guangdong, China, Pletholophus guangzhouensis sp. nov., is described based on morphological diagnostic features and molecular phylogenetics. The glochidia shells of the new species are subtriangular, medium-sized, and have a styliform hook on the ventral angle of each valve. Phylogenetic analyses based on the COI and 28S rRNA gene fragments indicated that Pletholophus guangzhouensis sp. nov. is the sister to Pletholophus tenuis + Pletholophus reinianus. The pairwise uncorrected COI p-distance analysis demonstrated genetic distances ranging from 5.27% (between P. guangzhouensis sp. nov. and P. tenuis) to 11.06% (between P. guangzhouensis sp. nov. and P. honglinhensis). Our findings suggest a significant underestimation of the diversity of freshwater mussel species in Guangdong. Further field collections and systematic studies are necessary to fully explore the biodiversity of this region. Furthermore, integrative classification methods and genetic research are essential for informing the development of effective conservation strategies.