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Alfalfa is the most important forage crop cultivated in semi-arid areas, both in rainfed and irrigated conditions. Water available for crops is a limited resource and improvement of water use efficiency (WUE) is an important goal for plant breeding. The objective of this study was to detect quantitative traits loci (QTL) for WUE in a mapping population of alfalfa. A F-1 mapping population was obtained and a genetic map based on 85 SSR markers was built. The F-1 plants and the parents were transplanted in soil columns, and WUE was measured during six regrowth cycles under well-watered conditions. QTL detection was carried out by single and multiple factor analysis of variance. The two parents significantly differed for WUE as expected. The F-1 population showed quantitative variation for this trait. In single factor ANOVA, nine markers/alleles had a significant effect on WUE variation. In multiple factor ANOVA, six markers/alleles had a significant effect and explained 31.0% of the variation. These QTL will be useful to better understand adaptation to water stress conditions in lucerne and to breed improved varieties.