Community assembly can change over time following variations in connectivity among habitats. Previous studies have demonstrated the influence of seasonal pulse floods on community structures by facilitating species dispersal, but the effects of short-term flash floods have been poorly investigated. We investigated fish community structures before and after a short-term flood in drainage ditches of paddy fields in the Kita River basin, Japan. The study sites were classified into three connectivity types, “Transient” (connected only during the flood, with a temporally-disappearing vertical gap between drainage ditches and downstream rivers), “Connected” (always connected, without the vertical gap), and “Disconnected” (always not connected, with the vertical gap). The abundance of Misgurnus anguillicaudatus, the only species that dispersed from paddy fields to drainage ditches, increased after the flood irrespective of the connectivity types, although the total abundance of the other species significantly increased after the flood only in the “Transient” sites. The dissimilarity of species composition between the (n-1)-th and n-th surveys at each site was consistently low in the “Connected” and “Disconnected” sites, whereas it rose just after the flood and gradually declined in the “Transient” sites. Species composition was significantly different among the connectivity types, indicating that some fish species need stable connectivity even during the non-flood period. The results showed that the short-term flood facilitated fish dispersal from paddy fields by the increased water discharge and that from downstream rivers by the temporary removal of vertical gap, emphasizing the importance of hydrological connectivity for biodiversity conservation in an agricultural landscape.