Brazil has one of the largest varieties of aquatic ecosystems and rich freshwater biodiversity, but these components have constantly been damaged by the expansion of unsustainable activities. Free-living nematodes are an abundant and ubiquitous component of continental benthic communities, occurring in all freshwater habitats, including extreme environments. Despite this, hardly any studies have examined the generic composition of nematodes in different latitudes and the geographic overlap of assemblages. We provide data on nematode genera from six regions in Brazil, over a north-south gradient spanning about 4000 km, encompassing rivers, coastal lakes, and reservoirs with different levels of human impact. Interpolation/extrapolation curves were generated and the zeta diversity was used to assess the overlap of nematode assemblages. Freshwater nematode assemblages comprised 54 families and 132 genera. Mononchidae, Monhysteridae, Chromadoridae, Tobrilidae and Dorylaimidae were the most diverse families. Differences in
diversity and high turnover of genera were found among regions, probably related to stochastic processes. Mononchus was the only widely distributed genus. Our results revealed a high biodiversity of free-living freshwater nematodes among the regions. The limited spatial coverage of the data reveals an enormous knowledge gap in a country with 12% of the world’s freshwater resources. The lack of spatial patterns, e.g., latitudinal variation, suggests that freshwater nematode assemblages are primarily structured by the intrinsic properties of habitats. This reinforces the uniqueness of freshwater ecosystems and suggests that the nematode assemblages may be sensitive to environmental disturbances, since the limited distributions of taxa may lead to lower resilience.