Abstract: Pleurostomatid ciliates, as a highly diverse and widely distributed unicellular eukaryote group, play a crucial role in the cycling of nutrients and energy in microbial food webs. However, research on pleurostomatids is insufficient, resulting in a paucity of molecular information and substantial gaps in knowledge of their phylogenetic relationships. In recent years, our team has investigated pleurostomatid diversity in various Chinese habitats including their systematic relationships and taxonomic circumscriptions which have been comprehensively analyzed using an integrative morpho-molecular approach. The results reveal that: (1) pleurostomatids can be categorized into two suborders: Protolitonotina subord. nov. and Amphileptina Jankowski, 1967; (2) Protolitonotina subord. nov. represents the ancestral pleurostomatid group and includes two genera, Protolitonotus and Heterolitonotus gen. nov. respectively, which are characterized by right kineties progressively shortened along rightmost full kineties and the absence of a left dorsolateral kinety; (3) Heterolitonotus gen. nov. represents an orphan lineage and is defined by an oral slit extending to its dorsal margin; (4) ‘Protolitonotus clampi’ does not group with congeners but instead represents an orphan lineage, thus Novilitonotus gen. nov. is established to which P. clampi is transferred as Novilitonotus clampi comb. nov.; (5) three new species, Apoamphileptus paraclaparedii sp. nov., Heterolitonotus rex gen. nov., sp. nov. and Loxophyllum apohelus sp. nov., are described; (6) helices 21es6a to 21es6d within V4 region of SSU rRNA may be used to discriminate pleurostomatids. The evolutionary relationships among all main lineages of pleurostomatids are discussed and a key to the identification of pleurostomatid genera is supplied.