This study aims to evaluate the dynamics of fish trophic guilds according to the longitudinal gradient of the Paraguay River, northern Pantanal, Brazil. Three river segments were sampled: plateau, confluence and plain. These segments have different physical and biological characteristics, with high water flow in forest areas in plateau and slow flow in meanders, with Pantanal typical vegetation. In total, 26,542 individuals distributed in 130 fish species were collected. The sampled species were characterized in seven trophic guilds. From the seven trophic guilds identified, only three were statistically related to the type of the environment; herbivores were more abundant in the plateau, piscivores in the confluence, and invertivores in the plain. According to values of corrected Akaike Information Criteria, the environmental variable that best explains the abundance of piscivorous fishes in the segments sampled in the Paraguay River was water transparency. For herbivores, the model that explained the variation in abundance was composed by temperature, altitude and dense forest proportion. The variable altitude best represented the abundance of invertivores. Water transparency, temperature, altitude, river width and dense forest proportion were determining factors for the distribution of piscivorous, herbivorous and invertivorous fishes as a response to an environmental gradient that meets its ecological requirements. Understanding the trophic relationships is fundamental for management actions, contributing to the maintenance of ecosystem services of different species. Therefore, future research must be taken into account regarding management and ecological relationships.