Ascites syndrome (AS) is a major cause of economic losses to the broiler industry. The tendency of broilers to develop AS was found to be heritable, suggesting that selective breeding could provide a solution to this problem. To further elucidate the genetic control of AS, AS-susceptible (AS-S) and AS-resistant (AS-R) lines were established by 3 cycles of divergent selection on pedigree data of AS mortality under AS-inducing conditions. The rapid divergence between the lines suggested the involvement of a major gene with dominance for AS resistance. It was hypothesized that the difference between the lines is controlled by a single dominant gene, denoted A, with AA (and some Aa) individuals in the AS-R line, and aa individuals in the AS-S line. The current study was designed to test this hypothesis by test-crossing heterozygous (Aa) sires from the AS-R line and F(1) from the AS-R x AS-S cross, with recessive homozygous (aa) dams from the AS-S line. A ratio of 1:1 was expected between progeny with AS vs. healthy progeny when reared under AS-inducing conditions. Test-cross progeny of 5 sires from the AS-R line segregated 1:1, indicating that these sires were heterozygous (Aa) in the suggested major gene and thus supporting the hypothesis of a single major gene with dominance of AS resistance. There was segregation among test-cross progeny of 8 F(1) sires, but with a 3:1 ratio of AS progeny to healthy progeny. The 3:1 ratio is expected if the F(1) sires are heterozygous (AaBb) with complementary interaction between the dominant alleles in 2 unlinked major genes. The segregation among test-cross progeny of the 9 heterozygous AS-R sires could also be explained by the same model. These results suggested that 2 major genes are responsible for the difference between the AS-R and AS-S lines. Resource populations derived from these lines will facilitate an efficient genomic search for these 2 genes. Once the alleles of these genes are identified and genotyping tests are developed, breeders will easily be able to select against AS susceptibility.