A genomic library from the commercial diploid cultivar 'Ouro' (Musa acuminata), enriched for CT- and GT-repeats, was used to isolate and characterize 23 microsatellite loci. These loci were tested in 10 Musa genotypes, representing various Musa genomic groups with distinct ploidy level. The number of alleles per locus ranged from one to seven, and 20 loci were highly informative. Four loci appeared to amplify B genome-specific alleles, while three loci seemed to be absent in the B genome. The polymorphism revealed by these loci will be extremely useful for genetic mapping, marker-assisted selection, germplasm characterization and evolutionary studies in Musa.