Freshwater ecosystems have experienced a great loss of biodiversity in the recent century due to eutrophication and loss of habitats, particularly in agricultural lowlands. Some of these ecosystems have recently been re-established, or entirely new lakes have been created to improve nutrient removal, biodiversity, and recreation. Fish have an important structuring role in lake ecosystems, but little is known about the temporal development of fish species richness or composition in new or re-established lakes, especially in comparison with natural lakes. We investigated the influence of environmental variables and landscape features on fish species richness and fish assemblage in drainage basins and in 34 new (between 0 and 99 years) and 193 natural lakes in Denmark, using structural equation modelling. Fish species richness in drainage basins is influenced primarily by basin elevation, lake area, and salinity at the basin outlet; low-salinity coastal regions are important migration pathways for many freshwater fish species, not just for anadromous or catadromous species. Land use does not appear to influence richness at the drainage basin scale, indicating that the influence of anthropogenic activities is minor or that significant effects thereof occurred a long time ago (decades to centuries). The drainage basin richness defines the apparent pool of species that may colonise lakes within the basin, but the actual number of species is particularly influenced by stream network connectivity. Disconnected lakes have fewer species, a deficit that is sustained over long time scales. In addition to the influence of stream connectivity, lake characteristics such as elevation, surface area, and water chemistry (alkalinity and pH) are also important predictors of fish species richness, whereas we did not find any effect of lake nutrient concentrations. The direct effect of land use was not evaluated. Ordination analysis shows that fish assemblage depends on both lake type (new/natural) and stream connectivity (connected/disconnected). Furthermore, species-specific occurrences reveal some differences between new and natural lakes and indicate that stocking plays a role in new lakes. While new and natural lakes may initially differ in terms of both how many and which species they contain, they converge over time in a process that can be enhanced by improving stream connectivity.