Two families of Helianthus lines, T− (low desiccation rate) and T+ (low desiccation rate), with contrasting osmotic adjustment (OA), obtained from a divergent selection programme, were compared for several morphological traits, ecophysiological parameters and molecular markers. The physiological analysis in a glasshouse experiment showed that the two families were identical in irrigated conditions except for a single trait, relative water loss (RWL: water loss rate of excised leaves). RWL could be used for these genotypes as a physiological marker of OA. A molecular analysis of the two contrasting lines, using differential screening of cDNA libraries, led to the isolation of three specific cDNAs, one of which, DRS26, showed high sequence homology with a mammalian amino-acid transporter. Subsequent RFLP and STS studies, using DRS26 and RAPD bulked analysis, enabled the identification of putative molecular markers of RWL and OA in T−[ratio ]T+ sunflower.