Understanding variations in species diversity and assemblage related with environments at a large spatial scale has been a central aim of community ecology for decades. The current study contributes to building the global perspective on the lake fish diversity and assemblage in China, and identifying the potential determinants, based on the multi-species approach fitted by Multivariate Regression Trees. A total of 425 fish species and subspecies recorded in 135 lakes across the mainland China were modelled with 19 potential environmental factors. Five fish assemblages were then patterned according to the similarity of fish composition and determinants. Diversity partition (alpha diversity and gamma diversity) was employed to compare the differentiation of fish diversity in Chinese lakes in multiple spatial scales. A significant difference between each assemblage was found: alpha diversity was lower in plateau lakes than in plain lakes. However, gamma diversity on plateaus was higher than any other regions. Altitude, minimum temperature of the coldest month, annual temperature range and precipitation during the driest month were the most important determinants affecting fish assemblages and diversity. Consequently, our findings inform the potential implications of fish species conservation and natural resources management.