The United States Department of Agriculture (USDA)—Agricultural Research Service (ARS)—National Clonal Germplasm Repository (NCGR) in Corvallis, Oregon, is a genebank that preserves strawberry genetic resources. Representatives of two Japanese diploid species, Fragaria iinumae Makino and F. nipponica Makino were collected for conservation by the NCGR during an expedition to Hokkaido, Japan. Fragaria iinumae may be a genome contributor to the cultivated octoploid strawberries. The objective of this study was to evaluate the genetic diversity of these two species by using simple sequence repeat (SSR) markers. Twenty of 82 Fragaria-derived SSRs, polymorphic among and within the two species, were selected for genetic analysis of 137 accessions. Genetic diversity, based on the proportion of shared alleles between the two species, in F. nipponica (0.4542) and F. iinumae (0.1808) was significantly different. Three wild interspecific hybrids were identified from intermediate memberships in the two diploid species groups revealed by using the clustering program, Structure. Principal coordinate analysis followed by non-parametric modal clustering (PCO-MC) grouped accessions into two clusters representing the two diploid species. Further clustering within the species groups generated with the program, STRUCTURAMATM, resulted in seven subclusters in F. iinumae and three in F. nipponica, which may represent breeding populations appropriate for clonal conservation. Long-term preservation of the species populations and the limited number of hybrids on the island is discussed relative to their geographical distribution and the geological history of Hokkaido Island.