The Pearl River Delta is the most economically active and densely populated region in South China. The production of domestic and industrial waste has increased considerably because of this economic development, and a 10% increase in waste production is estimated to have occurred in the past few years. The spatio-temporal patterns of phytoplankton species richness of the Pearl River Delta in 2012 were demonstrated in this study, based on data from 13 representative sampling sites covering most of the area of the river delta. During the investigation, 383 species from 7 groups were identified. The main groups were diatoms (Bacillariophyceae) and chlorophytes, accounting for 41.78% and 29.24% of the total numbers, respectively. Euglenophyta and Cyanobacteria were subsidiary groups. Physical and chemical variables were analyzed to assess key factors and their interaction mechanisms were demonstrated using redundancy analysis. The total species richness was significantly different among the stations in dry seasons, but not in wet seasons. In dry seasons, river water disturbance caused the suspension of diatoms. In wet seasons, exogenous supplementation occurred and the river became diluted, thereby causing a reduction in diatoms. The total species richness of the stations around Guangzhou and of several stations around the middle of the delta was higher than that of the other stations, which was mainly related to the spatial pattern of nutrient levels and the water exchange capacity. The spatial characteristics of different seasons showed that the species richness in dry seasons increased along the West River, the middle of the delta, and the area surrounding Guangzhou, indicating that nutrient contents were the most important factor. In contrast, in the wet seasons, the species richness on both sides of the delta was higher than that in the middle of the delta. This was influenced by water flow replenishment and water exchange capacity. Moreover, the proportional composition of various groups showed that diatoms accounted for the highest proportion of total phytoplankton species in the dry season, decreasing in the wet season. The proportion of diatoms decreased during floods, owing to the supplementation of chlorophytes, euglenophytes, and cyanobacteria through influx into the main river channel from dead zones. The percentage of diatoms in the Guangzhou station was significantly lower than that of the other stations. This was primarily because the Guangzhou station was located at the intersection of rivers, and there was algal supplementation by external rivers all year round, and secondly because the Guangzhou station was located in the center of the city. These areas have large water discharges, resulting in higher nutrient contents than other areas. On the basis of statistical analysis, we concluded that neutralization of exogenous supplementation and dilution of the river and the suspension of diatoms caused by river agitation not only affected the seasonal variations of species richness, but also the relative composition of different groups. The water exchange capacity and nutrient contents were the key factors determining the spatial distribution of species richness in the wet and dry seasons, respectively.