To facilitate the subgenome tracing in octoploid strawberries, pentaploid hybrids were developed by crossing diploid F. vesca (as female) with various octoploids. Genomic in situ hybridization using differentially labeled genomic DNA of F. vesca and F. iinumae was performed on a pentaploid hybrid. Fourteen chromosomes (seven presumably from F. vesca) preferentially took the F. vesca probe, and the remaining 21 either preferentially took the F. iinumae probe or had only a weak or absent hybridization signals. Initial analysis of meiotic pairing configurations in several pentaploid hybrids showed formation of multivalents at various stages from diplotene to metaphase I. Total “valent counts” (the numeric sum of all discrete chromosomal “objects”) at diakinesis provided a basis for assessing the degree to which homoeologous and multivalent associations are occurring. The finding that pollen mother cells mostly had less than 21 “valents” further suggested the existence of multivalent pairing. FISH analysis using 25S and 5S rDNA probes to mitotic and meiotic cells of pentaploid hybrids provided insight into meiotic pairing relationships in these pentaploids. A molecular karyotype, in which each single chromosome was differentiated based on FISH markers probed by rDNA and single-copy sequences, was constructed in F. vesca. Further application of molecular karyotyping would help tracing homo- or homoeo-logous chromosomes in pentaploid hybrids.