Phylogenetic relationships among 43 accessions of Fragaria, representing 14 species, and one accession of the outgroup Potentilla fruticosa, were assessed using nucleotide sequence data from the nuclear ITS region and from a region of the chloroplast genome including the trnL intron and the trnL-trnF spacer. Variability was low in both regions and lower in the chloroplast than in the nuclear sequences. The data were analyzed cladistically, separately and in combination. Greater phylogenetic resolution was obtained with the ITS and combined data than with the trn data; the strict consensus tree from the combined data analysis was very similar to that from the ITS analysis, but the position of some accessions was affected by the inclusion of the trn data. The results support the following conclusions: Fragaria iinumae is sister to all other species in the genus; F. vesca and F. nubicola are the diploid species most closely related to the polyploid species, including the tetraploid F. orientalis, the hexaploid F. moschata, and the octoploids F. chiloensis and F. virginiana; the latter two species are sister taxa; accessions of F. virginiana subsp. platypetala from Oregon and Nevada are more closely related to F. chiloensis than to other accessions of F. virginiana. The data provide support for the identification of some accessions in the National Clonal Germplasm Repository in Corvallis, Oregon.