Representational difference analysis (RDA) was initially used to identify differences between two inbred lines of chickens, line N and line 15I, on which the Compton mapping reference population is based. RDA was subsequently used to identify marker loci targeted specifically to chicken chromosome 16. Chromosome 16 contains the major histocompatibility complex (MHC), nucleolar organiser region (NOR) and Rfp-Y complex. To generate markers specific for this chromosome a bird was selected from the Compton mapping reference population which had inherited N line alleles for the MHC, NOR and Rfp-Y regions on this chromosome. DNA from this bird was compared with pooled DNA from 16 of its siblings, all of which had inherited line 15I alleles for the MHC, NOR and Rfp-Y regions. Initially amplicons were derived from BamHI digested samples, RDA products were cloned after the first round of hybridisation and 113 clones were investigated: 45 of these identified BamHI polymorphisms in this population. Of the 45 polymorphic clones, 17 have been mapped in the reference population so far, and these have identified seven new loci on chromosome 16. Interestingly a group of 16 other loci were linked on chromosome 4. The same birds were also compared by RDA following digestion with TaqI. Again large numbers of clones were generated of which 65 were investigated. Of these 17 clones were polymorphic and of five clones mapped so far three lie on chromosome 16. Two of the loci mapped to chromosome 16 have been used to identify yeast artificial chromosome (YAC) clones.