Trifolium species are native to the Mediterranean Basin where they are an important component of permanent pastures. A number of species have become naturalized in Australia and are agriculturally important in annual pastures. To understand the importance of genetic and ecotypic variation in the adaptation of Trifolium species to a new environment, seeds of three predominantly inbreeding and two predominantly outcrossing species of Trifolium were collected from 12 sites in Sardinia in 1998. Detailed ecogeographic information was collected at each site. The progeny were grown at the University of Western Australia Field Station at Shenton Park, Western Australia in 1999, and were scored for 10 morphological characters. Spatial analysis was applied to the data to improve the estimation of accession means, and to investigate the relationship with environmental variables that characterized the sites of collection. The spatially adjusted means were used as the basis for a principal components analysis. Ecogeographical factors at the sites of collection, particularly soil pH, are suggested to be more important than breeding system in determining the extent of genetic variation within the species. The species that showed the greatest genetic variation between accessions were the predominantly inbreeding species T. glomeratum and T. subterraneum, and the predominantly outcrossing species T. nigrescens. It is suggested that the wide genetic variation of these three species is largely due to their being in an ecogeographic environment close to their optimum, and to the possession of a mating system that is neither completely outcrossing nor completely self-fertilizing. The remaining two species, T. tomentosum and T. resupinatum, both occur more frequently on alkaline soils, and it is suggested that for the collected accessions of these two species the acid soils of Sardinia are a stressful environment that does not promote high levels of genetic variation.