Edible bananas are hybrids of various inter- and intraspecific combinations of Musa acuminata Colla and M. balbisiana Colla at different ploidy levels brought up by coexisting selection by farmers and nature. Our current study, based on a 461-bp-long sequence of the 5′ETS rDNA of 35 diploids and 19 cultivated varieties harboring 87 informative SNPs, revealed five groups of M. acuminata subspecies; A1: zebrina, A2: malaccensis, A3: burmannica/burmanicoides/siamea, A4: banksii/errans/microcarpa, A5: truncata, and a single cluster of B: M. balbisiana. We identified a new 5′ETS rDNA allele unidentified gene pool (UGP) absent in wild type accessions but present in diploid and triploid cultivars. This new allele is probably the result of recombination event in ancient ‘cultiwild’ populations. In the present study we observed a putative crossover event re-establishing a conventional allele. Genotyping triploid cultivars revealed that the Hom group cultivars are composed of two different ancestors while Cavendish Grande Naine, Cavendish Petit Naine, Cavendish Poyo and Gros Michel have three different ancestors. Hom Thong and Hom Taiwan were shown to have different ancestors compared to Gros Michel, unexpectedly even including subspecies truncata rDNA. We further improved our earlier established 5′ETS rDNA SCAR marker system by adding two new markers identifying M. acuminata subsp. truncata (Ridl.) Kiew and the newly identified UGP allele. We suggest that for breeding purposes, involvement of partially fertile diploid cultivar/cultiwild carrying the UGP gene pool may be an approach for developing new breeding programs and improving genetic studies of edible banana.