Abstract: The genus Cladophora Kützing is one of the largest genera of filamentous green algae (Cladophorales, Chlorophyta) and plays an important role in the aquatic ecosystem. The geographical distribution of Cladophora is cosmopolitan, from tropical to polar water, from freshwater to marine. On the one hand, Cladophora can provide important habitats for aquatic micro-animals and plants and be used in aquaculture and business as a valuable biological resource, they also can cause algal blooms in eutrophic waters frequently. Due to phenotypic plasticity of some morphological characters, such as branching pattern, filament dimensions and attachment organs, classification and identification of Cladophora have challenged taxonomists for years. With the development of technology, molecular methods demonstrate the effectiveness of Cladophora classification and reveal cryptic species diversity in several morphospecies. Although ribosomal DNA (rDNA) sequences are widely used in the phylogeny of Cladophorales, evolutionary analyses only based on single molecular marker, such as the small-subunit (SSU) rDNA or the large-subunit (LSU) rDNA, are difficult to resolve the phylogenetic relationships of Cladophora. An approach combining multiple molecular markers and increasing sample collection will be needed to arrive at a well-supported phylogeny. Chishui River, as a large and valuable tributary on the south bank of the upper Yangtze, has the feature of diverse landscape types and rich biodiversity. However, the diversity investigation of Cladophora in this area is still absent so far. In order to assess Cladophora diversity of Chishui River basin, a total of 38 sampling sites were established along the upper, middle and lower regions of the river. The field-collected filamentous samples were preserved in 10% formalin and 95% ethanol, respectively. These materials were taken back to the laboratory for DNA extraction and polymerase chain reaction (PCR) amplification. We sequenced three molecular markers including SSU rDNA, LSU rDNA and the internal transcribed spacer (ITS, ITS1-5.8S-ITS2) sequences to determine the phylogenetic position of these algae. All DNA sequence data generated for this study were submitted and deposited in GenBank. We used two datasets in the present study: dataset 1, a concatenated alignment of SSU+LSU from 80 cladophoracean samples (Chaetomorpha as an outgroup); dataset 2, a combined dataset of SSU+ITS+LSU from 51 freshwater Cladophora samples (plus 5 brackish Cladophora as an outgroup). Sequence matrices for phylogenetic analyses were aligned using MAFFT 7.0. All datasets were analysed using Bayesian Inference (BI) and Maximum Likelihood (ML). One thousand non-parametric boot strap replicates were inferred from 10 distinct alternative runs. Posterior probability values were calculated for 1×10⁶ generations, with sampling conducted every 1000 generations. The results showed that: (1) Cladophora diversity is higher in Chishui River Basin, where the widespread species C. glomerta and the rare species C. rigida both distributed. Cladophorales (including Cladophoraceae and Pithophyceae) samples were obtained at a total of 21 sampling sites, 19 of which existed Cladophora samples. The distribution of these algae covered the upper, middle and lower regions of Chishui River, at the same time, Cladophora species diversity showed a decreasing trend from upstream to downstream. (2) Sequence analysis showed that the current freshwater Cladophora taxa contained at least ten phylogenetic clades, six clades of which contained samples collected from this area. These six clades are as follows: clade 1, clade 2, clade 4, clade 7, clade 9 and clade 10, respectively. (3) The phylogeny based on SSU+ITS+LSU divides freshwater Cladophora into two major groups (group Ⅰ and group Ⅱ). Clade 1, clade 2 and clade 4 formed a group I, as did clade 5, clade 6, clade 7, clade 9 and clade 10 (group Ⅱ). Compared with the phylogeny based on SSU+LSU, the support value of each clade (except clade 4) of the phylogeny based on SSU+ITS+LSU is higher (BI=1.00; BP＞95), which further supported that using multiple molecular markers, and particularly, adding the rapidly evolving molecular marker ITS, are useful and effective in analyzing the taxonomy and phylogeny of Cladophora. Due to the lack of typical morphological characteristics, the species names of most clades are not determined for the time being, except clade 1 (C. glomerta) and clade 9 (C. rigida). Notably, C. rigida, differed from other freshwater Cladophora, had some unique characteristics, such as being unbranched, with a large cell diameter (180—429 µm) and smaller length: diameter (L﹕D) ratio (0.5—3.3) than other species. This species is now only reported in Chishui River basin and Hubei Province of China.　　In conclusion, our results not only revealed the Cladophora species diversity of Chishui River based on multiple molecular markers, but also enriched the classification of freshwater filamentous green algae of China, as well as provided basic data for the ecological environment protection of Chishui River. Next, continued investigation and exploration of other regions of China that have not yet been covered will be needed, which likely yield additional records and findings.