Water pollution has been a significant issue in the Huai River Basin (HRB) of China since the late 1970s. In July and December 2013, two field investigations were carried out at 10 sites along the main streams of the basin. The monitoring indices contained both physicochemical variables and the structure and composition of phytoplankton communities. The correlations between communities and physicochemical variables were analyzed using cluster analysis and redundancy analysis. Moreover, water quality was evaluated using the comprehensive nutrition state index (TLI) and Shannon-Wiener diversity index (H). Results indicated that more phytoplankton species were present in December than in July, but total density was less in December. Phytoplankton communities in the midstream of the Shaying River were affected by the same physicochemical factors throughout the year, but ammonia nitrogen and total phosphorus had the greatest influence on these sites in July and December, respectively. The water pollution status of the sampling sites was much greater in the Shaying River midstream than at other sites. TLI was more suitable than H for assessing water quality in the study area. These results provide valuable information for policy makers and stakeholders in water quality assessment, water ecosystem restoration, and sustainable basin management in the HRB.

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