Chicken B-G-subregion cDNA probes were used to analyze restriction fragment length polymorphisms (RFLP) of the B-G subregion of the chicken major histocompatibility complex. Genomic DNA from chickens representing 17 of the 27 standard B haplotypes were digested with restriction endonucleases and analyzed in Southern hybridizations with two cDNA clones from the B-G subregion. Each B-G genotype was found to produce a unique pattern of restriction fragments in these Southern hybridizations. With 15 of the 17 genotypes examined, the different genotypes could be readily distinguished in hybridizations produced with DNA digested with a single restriction enzyme, PVU II. The two additional genotypes produced nearly identical patterns in PVU II preparations and with three additional enzymes as well, but were readily distinguishable in Eco RI digestions. For many of the haplotypes, samples from several individuals in different flocks were examined. In every instance, genotyping by RFLP pattern was found to confirm the B-G allele assigned serologically.