This dissertation describes the application and development of molecular tools that have great potential for use in studying variation in strawberry germplasm. The first study evaluated 91 microsatellite (simple sequence repeat, SSR) markers for transferability in 22 Fragaria species and their utility in fingerprinting octoploid strawberries. Out of the transferable markers, a reduced set of four SSRs was developed based on polymorphism, multiplexing ability, reproducibility and ease of scoring. Unique SSR fingerprints were obtained for over 175 Fragaria samples representing 22 Fragaria species used in the study. Testing of two molecular markers linked to the red stele and anthracnose resistances identified potential sources of resistance in previously untested genotypes. Further characterization of these accessions is warranted to validate resistance and usefulness in breeding. In the second study, 20 SSRs polymorphic in wild Asian diploids, F. iinumae and F. nipponica, from Hokkaido, Japan were selected for genetic analysis of 137 accessions from 22 locations. Principal coordinate analysis followed by non-parametric modal clustering grouped accessions into two groups representing the two species. Further clustering within the groups resulted in 10 groups (7-F. iinumae, 3-F. nipponica) that suggest lineages representative of the diversity in Hokkaido, Japan. The third study tested plant DNA barcodes, the nuclear ribosomal Internal Transcribed and the chloroplast PsbA-trnH spacers, for Fragaria species identification. The 'barcoding gap', between within species and between species variation, was absent and prevented identification of Fragaria species. The fourth study evaluated the genetic diversity of 94 accessions representing 22 Fragaria species using four universal chloroplast SSR loci. Genetic diversity was moderate (0.54) despite the homoplasy observed. Species-specific haplotypes for F. nipponica, F. orientalis, F. iinumae and F. nilgerrensis were identified. Sequencing whole chloroplast genomes using Illumina in a final study revealed a close maternal genome relationship between F. vesca ssp. bracteata and the octoploid species supporting a North American origin of the octoploids and the polyphyly of F. vesca. Calculation of divergence time of Fragaria revealed young evolutionary age of the genus at 2.7 million years and of the octoploids at 450,000 years
