We investigated the influence of environmental factors in fish communities of 146 palustrine wetlands, covering a wide range of altitude and wetland surface area in Neotropical region. Two questions were analyzed: (1) Are wetland altitude, area, habitat diversity, hydroperiod (permanent and intermittent), ecoregion, and macroinvertebrate richness good predictors of occurrence, richness, abundance and composition of fish species? and (2) Are the predictors of fish richness similarly applicable to different ecoregions in Southern Brazil? Our data showed that fish richness was related to habitat diversity and macroinvertebrate richness, and fish occurrence was influenced by wetland area and macroinvertebrate richness. Fish abundance was influenced by altitude, hydroperiod and macroinvertebrate richness, and the fish composition was jointly associated with ecoregion, and hydroperiod. The predictors of fish richness were not similarly applicable to different ecoregions. Our results showed that the habitat diversity, macroinvertebrate richness, altitude and hydroperiod were the environmental predictors that potentially structure and maintain the fish occurrence and richness in southern Brazil palustrine wetlands. Such information is essential to develop wetland conservation and management programs in this region, where more than 90 % of wetland systems have already been lost and the remaining ones are still at high risk due to the anthropogenic activities.