Two diploid species of hybrid origin, Argyranthemum lemsii and A. sundingii, have been described from different valleys in the Anaga peninsula, north-east Tenerife. They have previously been shown to originate from hybridization between the same parental species, the montane A. broussonetii and the coastal A. frutescens, A. broussonetii being the chloroplast donor in one valley and A. frutescens in the other. The specific status of the two hybrid species has been questioned. In this study we used karyotype analysis, FISH, and GISH to address the question of multiple diploid hybrid speciation. GISH did not discriminate clearly between the parental genomes, but differential labelling was observed in separate hybrid populations, indicating different chromosomal rearrangements in different valleys. Small karyotype differences and local loss of rDNA were also observed. Thus separate origins of the same hybrid combination in different valleys in Tenerife have been verified. Our results add some support to the recognition of two species of hybrid origin, but the case serves to illustrate some of the many problems connected with the species concept in plants. © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society, 2003, 141, 491–501.