The Pearl River, which is 2400 km long and flows into the South China Sea, is the longest river in subtropical southern China. The warm humid climate of the region, which promotes high species diversity, has resulted in significant aquatic biodiversity and the development of lucrative commercial fisheries. Nevertheless, studies on the spatial distribution of fish communities in the Pearl River system are limited, especially, considering the decline in global fishery resources due to numerous anthropogenic stressors, such as overfishing, dam construction, introduction of invasive species, and climate change. Thus, studies on the spatial distribution of fish species in the Pearl River are urgently needed. This study investigated the spatial patterns of fish diversity and distribution in the Pearl River Basin based on an investigation of 13 sample sites. A total of 10,119 individual fishes were collected during the study period, comprising 94 taxa representing 17 families and 72 genera. The Cyprinidae family, representing 49 taxa, accounted for 52% of the total number of fish species collected. Cyprinidae were the dominant family in the Pearl River, followed by Bagridae (7%; seven species) and Cobitidae (4%; four species). Non-metric multidimensional scaling (NMDS) was applied to the collected data and three separate groups were identified. The spatial variation in upstream fish assemblages differed significantly from that in downstream assemblages (P < 0.05), although clustering and diversity showed a significant spatial autocorrelation. A significantly high species diversity was recorded in the Pearl River, but low species diversity existed in the up and downstream reaches. Redundancy analysis was used to describe the relationship between the diversity patterns of fish species and associated environmental factors. Temperature, precipitation, water discharge, river width, and water chemistry play important roles in species diversity, with the average annual temperature accounting for the largest proportion of variance. A comparison with historical data for the Pearl River revealed a dramatic decline in fish species and a significant change in the spatial distribution of fish communities. Currently, the most serious threat to the ecology of the Pearl River network is the invasion of alien species. For example, non-native tilapia are now widely distributed in most of the Pearl River system, even surviving in the cooler, upper reaches of the Nanpanjiang River. In the Hongshuihe, Beijiang, and Dongjiang Rivers, tilapia is the dominant species recorded and has frequently been collected in the middle and lower reaches of the Pearl River. The present study is part of a long-term investigation into the wild fishery resources of the Pearl River. Therefore, understanding the processes outlined in this study will assist in the conservation of fish community diversity, which is critical to the success and sustainability of commercial fisheries in the Pearl River. Regularized discriminant analysis (RDA) is used by ecologists to relate a dataset (Y) of response variables (such as species abundance) to a second dataset (X) of explanatory variables (often environmental factors) and consistently outperformed other statistical tests using all the data in this study.