The genome composition of the octoploid, cultivated strawberry awaits rigorous delineation. Initial insight into the organization and content of strawberry gene space comes from analysis of 50 genomic (fosmid) library inserts totaling ~1.75 Mb from the ~200 Mb genome of the diploid model species Fragaria vesca. Intergenic distances were generally low (0.5 to 5 kb), allowing for amplification of PCR products spanning highly polymorphic intergenic regions. Allele mining at such “gene pair” loci offers a new window onto allelic diversity and genome compositions of octoploids F. ×ananassa, F. chiloensis and F. virginiana. Three distinct haplotype classes were detected at the gRGA1-Subtilase gene pair locus in F. virginiana, providing molecular evidence that its genome is derived from at least three distinct diploid ancestors. Considerable variation was also present within haplotype classes, supporting the expectation that up to eight haplotypes can be differentiated at a single locus in an octoploid individual. A diploid (F. vesca) × octoploid (F. virginiana) cross has yielded a segregating pentaploid population, which is being used to assess patterns of haplotype transmission from the octoploid parent.