For understanding the reproductive dynamics of fish assemblages and their relationship with environmental conditions, we need to know more about spawning areas and periods. Studies on temporal and spatial patterns of occurrence of fish eggs and larvae are important for obtaining this kind of information quickly and accurately. In this study, we analysed spatial and temporal patterns of fish larvae in different biotopes in a stretch of the floodplain of Upper Parana River, in Ilha Grande National Park, southern Brazil. We carried out night sampling during six spawning periods, from 2001 to 2008. We selected 18 sampling sites distributed in the mainstream, tributaries and lagoons, encompassing several types of biotopes. We observed significant spatial and temporal differences among biotopes in the composition and structure of the fish larvae assemblages. Lentic environments favoured the development of sedentary and migratory species, whereas tributaries were used as migratory routes and spawning areas. The assemblage in period I differed from the assemblages of other periods, probably because of the presence of dams in the Parana River. No association was observed between environmental variables and spatial and temporal patterns of the ichthyoplankton assemblages. Hence, conservation measures are essential to maintain the environmental integrity of spawning (tributaries) and development areas (lagoons) for the maintenance of biodiversity in the floodplain of Upper Paraná River.