Strawberry is one of the five fruit crops included in the USDA-funded multi-institutional and trans-disciplinary project, “RosBREED: Enabling Marker-Assisted Breeding in Rosaceae”. A Crop Reference Set (CRS) was developed of 900 genotypes and seedlings from 40 crosses representing the breadth of relevant diversity and encompassing founders used in breeding the domesticated strawberry. Individual native species and cultivar genotypes were included along with 10 progeny from 36 of the crosses of genotypes representing eastern and western North American and European short day and remontant cultivars. This CRS has been phenotyped in five U.S. states. Over 14 fruit quality traits have been studied, as well as remontancy, truss size, peduncle length, crop estimate, plant architecture, and disease resistance. The phenotyping conducted in the first growing season showed considerable variability amongst the genotypes and the locations for all of the characteristics. General and specific combining ability variance components were determined from the populations in order to provide breeders with guidance on the most effective breeding strategies for incorporating the superior traits from this germplasm into their programs.