<p>The roles of lake area, depth, and isolation in regulating species richness and ecological structure of floodplains is not well understood. We investigated these roles for fish assemblages of the Upper Paraná River floodplain (southern Brazil), comparing native and non-native species and using taxonomic and functional approaches. This study surveyed lakes from 2010 to 2011, obtaining 152 seine net samples and measuring limnological variables. Metrics examined included abundance, richness, and functional traits. The lake area was positively related to species richness but only after accounting for lake depth, which had a stronger effect on fish metrics. Both the richness of native and non-native species and the abundance of native species decreased with lake depth, which was also negatively related to chlorophyll concentration and turbidity. The abundance of non-native species was higher in isolated lakes, suggesting favorable conditions for their establishment, potentially due to reduced predation and suitable reproduction habitats. Both native and non-native species in isolated lakes showed a higher proportion of species with reproductive strategies requiring minimal parental care. This suggests that isolated lakes may function as natural nurseries for fish, offering optimal conditions for egg laying and development. These findings highlight the need to include non-native species in floodplain ecosystem management strategies, such as monitoring programs and measures to prevent the spread of invasive species. Grouping all species together may overlook these important nuances. Conservation efforts should address the complex interplay between environmental variables and anthropogenic interventions to ensure the long-term sustainability of these ecosystems and the diverse fish communities they support.</p>