Abstract – A population of the exotic pest fish Gambusia holbrooki inhabiting a drainage channel was sampled regularly to record responses to flooding and subsequent population reestablishment. The flood reduced numbers in the channel to near-zero levels. After remaining very low for 2 months, densities increased steadily through juvenile recruitment and the concentration of fish in drying pools. Tagging revealed that in general, movements between pools separated by only a few metres were relatively limited. There was evidence for habitat segregation and population subdivision, as fish from different pools varied markedly in terms of mean population density, movement behaviour, sex and size composition and juvenile recruitment. The most favoured pool was relatively deep, well lit and had the greatest habitat diversity. Gambusia control measures are likely to be most effective if timed to coincide with floods (which reduce local population densities) and/or droughts (which concentrate fish and allow targeting of source populations).