Salmonella enterica serovar Enteritidis is a gram-negative bacterium that negatively affects human and animal health. Many eukaryotes use antimicrobial peptides (α-defensins, β-defensins, γ-defensins, and cathelicidins) in innate immune responses to fight bacterial infections. Poultry gallinacins are the functional equivalents of mammalian β-defensins. Two related advanced intercross lines of chickens were analyzed for association of gallinacin genotypic variation with Salmonella Enteritidis burden levels in the cecum and spleen after infection. Thirteen genes of the chicken β-defensin cluster (GAL1–13) were sequenced from individuals of each advanced intercross line, plus the founder broiler sire and representatives of the highly inbred Leghorn and Fayoumi founder dam lines. The mean was 17 single-nucleotide polymorphisms (SNPs) per kilobase. One single-nucleotide polymorphism per gene was genotyped with SNaPshot to test for statistical associations with Salmonella Enteritidis colonization after challenge. Among the 13 gallinacin genes evaluated, the single-nucleotide polymorphisms in all genes in a cluster of three adjacent genes (GAL11, GAL12, and GAL13) were associated with bacterial load in the cecal content in the broiler × Leghorn advanced intercross line (three-gene SNP genotype effect, P < 0.008). The results strongly suggest a role of the gallinacins in defense of poultry against enteric pathogens. The use of gallinacin single-nucleotide polymorphisms as molecular markers for genetic selection for Salmonella Enteritidis resistance might result in reduced bacterial burden via development of an enhanced innate immune response.