Concerns over yield declines in bananas and plantain^ uue to the spread of Black Sigatoka disease in Musa have drawn attention to the collection of Musa germplasm and its use in conventional and biotechnological improvement programs. This report demonstrates the use of chloroplast DNA (cpDNA) restriction fragment length polymorphisms (RFLPs) for differentiating cytoplasms of various Musa clones. DNA was extracted from lyophilized leaf blade tissue and digested with either Eco RI, Hind III, Bam HI or Pst I. Southern blots onto nylon membranes were probed using radioactively labeled heterologous orchid and lettuce cpDNA fragments. Among the 14 Musa clones examined, a single M. balbisiana and four M. acuminata-type cytoplasms were differentiated. The ability to distinguish between cytoplasms and to place plants within a cytoplasmic grouping demonstrates the usefulness of RFLP technology in evaluating diversity and determining the ancestry of Musa clones.