The cellular receptor of subgroup B avian leukosis virus (ALVB) is encoded by a gene at the tumour virus B (TVB) locus. TVB alleles encode specific receptors permitting infection by exogenous ALVB or avian leukosis virus subgroup D (ALVD) as well as endogenous avian leukosis virus subgroup E (ALVE), and thus susceptibility is dominant to resistance. Two single nucleotide polymorphisms at the TVB locus have been reported distinguishing three TVB alleles (TVB*S1, TVB*S3 and TVB*R). We have developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay using the two single nucleotide polymorphisms to define three observed allelic haplotypes and to identify the six possible TVB genotypes consisting of the three haplotypes in defined laboratory strains of chickens. One additional potential allelic haplotype and four genotypes were also briefly discussed. Chickens from parents heterozygous for different TVB alleles were challenged with Rous sarcoma viruses of subgroup ALVB and ALVE to induce wing-web tumours. Tumour incidences were evaluated between chickens of the genotypes determined with this newly developed PCR-RFLP assay. Importantly, chickens typed with this assay as TVB*S3/*S3 were resistant to infection by ALVE only, and those TVB*R/*R were resistant to both ALVE and ALVB. Furthermore, a vast majority of chickens with the susceptible TVB*S1/- genotypes developed a tumour. This PCR-RFLP assay enables a relatively rapid assessment of all six anticipated TVB genotypes in experimental strains of chickens undergoing segregation for TVB*S1, TVB*S3, and TVB*R alleles. This non-infectious assay should be further evaluated for the capacity to select and breed commercial chickens for genetic resistance to infections by ALVB, ALVD and ALVE.