Functional genes were selected for linkage analysis mapping using the East Lansing (EL) reference population ¿[Jungle Fowl (JF) x White Leghorn (WL)] x WL¿. The approach used was based on the identification of DNA sequence polymorphisms in the introns of those genes found in JF and WL. Deoxyribonucleic acid sequence analysis revealed single base substitutions in introns of six Type I marker genes: adenylate kinase 1 (AK1), aldolase B (ALDOB), a lysosomal membrane protein gene (LAMP1), vitellogenin 2 (VTG2), apolipoprotein A1 (APOA1), and creatine kinase B (CKB). Transitions or transversions were found in introns of AK1, ALDOB, LAMP1, VTG2, APOA1, and CKB. A transversion in the intron of the JF allele of AK1 generated a unique BspHI cleavage site. The design of polymerase chain reaction (PCR) primers based on the site of base substitution led to the specific amplification of the JF allele in the remaining five genes. A size polymorphism in the PCR production derived from iron response element binding protein (IREBP) distinguished the JF from the WL allele. Linkage analysis of the EL reference population revealed that these candidate genes were located in the following EL linkage groups (E) or chromosomes (Chrom) of the chicken genome: AK1 (E41), VTG2 (E43), APOA1 (E49), CKB (E07), LAMP1 (E01), ALDOB (Chrom Z), and IREBP (Chrom Z). Provided that a base substitution can be found in the parents of the reference population, this PCR-based approach can be used to map any cloned candidate gene. This approach will lead to further information on synteny of the chicken genome with cognate genes of mammalian species.