The sex-linked barring gene of the chicken (Gallus gallus), first identified in 1908, produces an alternating pattern of white and black bars in the adult plumage. More recent studies have shown that melanocytes in the developing feather follicle of the Barred Plymouth Rock experience premature cell death, whereas initially it was thought that melanocytes remained viable in the region of the feather devoid of pigmentation but were simply inhibited from synthesizing melanin. In an attempt to reconcile these 2 different hypotheses at the molecular level, we have taken a gene mapping approach to isolate the sex-linked barring gene variant. We developed a mapping population consisting of 71 F2 chickens from crossing a single Barred Plymouth Rock female with a White Crested Black Polish male. Existing and novel microsatellite markers located on the chicken chromosome Z were used to genotype all individuals in our mapping population. Single marker association analysis revealed a 2.8-Mb region of the distal q arm of chicken chromosome Z to be significantly associated with the barring phenotype (P<0.001). Further analysis suggests that the causal mutation is located within a 355-kb region showing complete association with the barring phenotype and containing 5 known genes [micro-RNA 31 (miRNA-31), methylthioadenosine phosphorylase (MTAP), cyclin-dependent kinase inhibitor 2B (CDKN2B), tripartite motif 36 (TRIM36), and protein geranylgeranyltransferase type I, beta subunit (PGGT1B)], none of which have a defined role in normal melanocyte function. Although several of these genes or their homologs are known to be involved in processes that could potentially explain the barring phenotype, our results indicate that further work directed at fine-mapping this region is necessary to identify this novel mechanism of melanocyte regulation.