Employing the flow-sorted chromosome 20-specific DNA library LL 20 NS01, we isolated seven novel unique poly- and monomorphic DNA markers specific to human chromosome 20. Initially, 201 phage clones were analyzed regarding insert size and repetitivity. By testing 14 single- and low-copy number clones for their ability to detect RFLPs, three polymorphisms were revealed by two probes, pFMS22-1.4 [D20S22] and pFMS76 [D20S23]. Seven of twenty probes (35%) were assigned to chromosome 20 using a somatic cell hybrid DNA panel. Five of them were regionally mapped by in situ hybridization. Three DNA markers, pFMS51 [D20S29], pFMS76 [D20S23], and pFMS106 [D20S30], were assigned to 20p11.2–p12, and two markers, pFMS22-1.4 [D20S22] and pFMS135 [D20S31], to 20q12–q13.3. Our new chromosome 20-specific DNA markers should be useful for the molecular characterization of this rather underpopulated human chromosome.