The persistence and specificity of fish host-microbial interaction during evolution is an important part of exploring the host-microbial symbiosis mechanism. However, it remains unclear how the environmental and host factors shape fish host-microbe symbiotic relationship in subtropical rivers with complex natural environments. Freshwater fish are important consumers in rivers and lakes and considered keystone species in maintaining the stability of food webs there. In this study, patterns and mechanisms shaping gut microbiota community in 42 fish species from the Pearl River in the subtropical zone of China were investigated. The results showed that fish host specificity is key driver of gut microbiota evolution and diversification. Different taxonomic levels of host showed different degrees of contribution in gut microbiota variation. Geographical location and habitat type were the next most important factors in shaping gut microbiota across the 42 fishes, followed by diet and gut trait. Our results emphasized the contribution of stochastic processes (drift and homogenizing dispersal) in the gut microbial community assembly of freshwater fishes in the middle and lower reach of the Pearl River. Phylosymbiosis is evident at both global and local levels, which are jointly shaped by complex factors including ecological or host physiological filtration and evolutionary process. The core microbiota showed co-evolutionary relationships of varying degrees with different taxonomic groups. We speculate that host genetic isolation or habitat variation facilitate the heterogeneous selection (deterministic process), which occurs and results in different host-core bacterium specificity.