Novel ecosystems emerge from alterations to historic abiotic regimes and contain new species combinations. Everglades canals offer an opportunity to understand the function of novel habitat for native and non-native fishes and how novel conditions in turn influence distribution, abundance and assembly patterns. I examined native and non-native fish assemblages collected across a gradient in novelty, defined by the loss of wetland connectivity and habitat complexity. As novelty increased, native species richness and abundance strongly declined, and the contribution of non-natives increased. Community structure vastly differed among canals and was strongly influenced by spatial factors and secondarily by hydrological factors. Natives and non-natives had opposing responses to key hydrologic and habitat parameters. This study represents the first comprehensive assessment of Everglades canal fishes, providing insight into the factors influencing native and non-native abundance and assembly patterns and contributing to our understanding of this novel but permanent habitat.