Jiménez‐Segura LF, Palacio J, Leite R. River flooding and reproduction of migratory fish species in the Magdalena River basin, Colombia. Ecology of Freshwater Fish 2010: 19: 178–186. © 2010 John Wiley & Sons A/S Abstract – In most tropical rivers subject to a single‐yearly flooding, migratory fish usually spawn in the onset of the flooding and larvae drift to their nursery habitats. To define when the migratory fish species reproduce, its relationship with the water level and when nursery areas in floodplain lakes are supplied with ichthyoplankton in the Magdalena River, a two‐yearly peak flooding river, we sampled the ichthyoplankton weekly throughout 2 years. The fish species Pseudoplatystoma magdaleniatum , Prochilodus magdalenae , Leporinus muyscorum , Sorubim cuspicaudus and Curimata mivartii spawn twice a year in the main channel and its larvae input into the floodplain lakes are not always associated with flooding. Spawning in the main channel and larvae inputs into the floodplain lakes suggest that the habitat used by the migratory fishes may be consistent with some hypotheses on habitats used by these fishes.