River-floodplain ecosystems are in delicate balance and are impacted by even minor changes in water availability. In this study, we surveyed fish assemblages and investigated environmental and landscape parameters in a total of 135 floodplain waterbodies (rivers, diversion canals, ponds, irrigation ditches, paddy fields, and wetlands) in the Chao Phraya River Basin in rainy (September 2014) and dry (March 2015) seasons. Factors affecting fish species richness in each type of waterbody were analyzed using generalized linear mixed models. Floodplain area around each surveyed waterbody was a major factor determining fish species richness in rivers, diversion canals, and ponds in the region. The contribution of floodplain area was equivalent to that of hydrology (current velocity, water depth) and water quality (dissolved oxygen, turbidity) in the waterbodies. The population of juvenile fishes was increased in temporarily connected floodplain waterbodies to main rivers compared with isolated waterbodies, and fluvial and lacustrine fishes were observed in the temporary inundated floodplain waterbodies during the rainy season. The high dependence of fish species richness on floodplain area in the region appeared to be a result of the use of inundated floodplains by fish species to forage and breed. Our results highlight the impact of flood control measures that reduces floodplain area. These measures must be reviewed to ensure the conservation of fish biodiversity in the Chao Phraya River Basin, one of the world’s most threatened floodplain systems.