Abstract: Poterioochromonas malhamensis is a typical mixotrophic single-celled flagellate. Due to its small size and variable morphology, the correct identification of this flagellate is still challenging. So far, there is no detailed morphological description for the species. In this study, one algae-grazing flagellate from Chlorella mass culture was isolated, which was identified as P. malhamensis CMBB008. Through fluorescence dying, scanning and transmission electronic microscopy, the morphological features were described in detail. In addition, SSU rDNA and rbcL gene sequences were also obtained to investigate phylogenetic position of this species. Based on previous morphological description, the morphology of lorica and chloroplast was documented: lorica length varied between 18.3—47.5 μm, the cup width was 8.5—11.3 μm, the cup depth was 6.3—10.7 μm, and the strain has one or two connected bilobed chloroplast. Using light and scanning electronic microscopy, siliceous cyst was firstly reported: a unique trilaminar collar and plug on the top. In addition, some primary organelles and metabolite such as oil droplets and chrysolaminarin vacuole were observed. Comparing different species and strains, the results revealed that lorica and chloroplast, which were regarded as important features of Poterioochromonas, were not suitable for identification of P. malhamensis because of high variable morphology. However, the cyst morphology was a candidate feature for species differentiation in Poterioochromonas. Molecular phylogenetic analysis showed that all strains of Poterioochromonas clustered in a clade supporting the genus was monophyly. P. malhamensis and P. stipitata formed sister clades in big clade of Poterioochromonas, and clade Poterioochromonas also divided in two sister clades, in which CCMP3181 probably be a species different from P. malhamensis. The study provides complete morphological and molecular data for P. malhamensis and valuable reference for the genus of Poterioochromonas in morphology and phylogeny.