Using AFLP fingerprinting, the genetic variation in a collection of 94 H. rosa-sinensis cultivars was assessed, and their phylogenetic relationships with other Hibiscus species was determined. All eight AFLP primer combinations (PCs) produced informative marker bands, and the use of four PCs was sufficient to uniquely differentiate each accession. Three major clusters were identified: group A included 87 of the 94 H. rosa-sinensis cultivars, including within it two sub-clusters related to flower shape and size; group B included seven old H. rosa-sinensis cultivars together with H. storckii, considered to be their progenitor; group C included H. denisonii, H. kokio. H. genevii and H. liliiflorus which, together with H. boryanus, H. arnottianus and H. schizopetalus, are thought to have contributed to the wide genetic variability characteristic of H. rosa-sinensis. The lowest level of genetic similarity with H. rosa-sinensis was shown by H. tiliaceus, H. syriacus, H. cannabinus and H. panduriformis, all of which are sexually incompatible with H. rosa-sinensis and thus are not likely to have been implicated in its origin. Our results demonstrated that AFLP fingerprinting provides a valuable means of assessing genetic variation within H. rosa-sinensis and elucidating its phylogenetic relationship with other Hibiscus spp.