Three Diophrys species, D. quadrinucleata n. sp., D. oligothrix Borror, 1965 and D. scutum (Dujardin, 1841) Kahl, 1932, were identified from the subtropical brackish waters in China, their morphology was investigated using live observation and protargol impregnation. D. quadrinucleata n. sp. can be distinguished from the congeners by four ellipsoidal macronuclear nodules. Phylogenetic analyses show that the three species with single marginal cirrus are closely related, namely D. quadrinucleata n. sp., D. apoligothrix Song et al., 2009 and D. japonica Hu, 2008. Our population of D. oligothrix corresponds very well with previous populations in both living morphology and ciliature. However, the SSU rDNA sequences of three well-described populations differ from each other by 11 to 18 nucleotides, indicating that there might be cryptic species in D. oligothrix. Moreover, we provided a detailed description of the symbiotic bacteria/archaeobacteria (?) concentrated on the back surface of D. scutum based on the present population, which was either overlooked or misinterpreted as cortical granules previously.