The D20S6 locus has been sublocalized by in situ hybridization using the pD3H12 probe to human chromosome band 20p12 and the D20S4 locus using the pMSl-27 probe to 20q13.2. A rare new restriction fragment length polymorphism detected in <i>Msp</i>I-digested DNA by the pMSl-27 probe is reported. Linkage studies in nine families have shown that the D20S6 locus is linked to D20S5 (formerly mapped to 20p12 by in situ hybridization) with a maximum likelihood estimate of 0.07 for the recombination frequency (lod score = 9.07) and a confidence interval of 0.02 to 0.14. Estimated recombination frequencies were similar in males and females. Using both two- and multipoint analyses, linkage of D20S4 with the D20S5 and D20S6 loci was excluded and the suggested order for the three loci on chromosome 20 is D20S5-D20S6-centromere-D20S4. D20S5 and D20S6 are very useful markers for linkage studies because of their close proximity and reasonably good polymorphic information content values.