This is the first report of targeted development of B genome microsatellite markers in Musa. A total of 44 sequences with microsatellites were isolated from an enriched library of Musa balbisiana cv. ‘Tani’ (BB genome). Of these, 25 were polymorphic when screened on 14 diverse diploid and triploid Musa accessions. The number of alleles detected by each marker ranged between one and seven. All 25 microsatellite markers generated amplification products in all species and genome complements. These new microsatellite markers fill an important gap for diversity assessment and linkage mapping studies in plantain (AAB) and cooking banana (ABB).