ABSTRACT Pullorum disease is a bacterial disease caused by Salmonella pullorum in chickens, which is characterized by gastrointestinal infection and diarrhea. In traditional perspectives, research on pullorum disease primarily focused on clinical symptoms, epidemiological characteristics, and the pathogenic sites. This study, however, approaches the subject from the standpoint of host genetic basis and gut microbiota. For the positive and negative offspring chicks, which are the offspring of positive roosters and hens and negative roosters and hens, respectively, we conducted whole-genome association analysis and identified 195 SNPs and 79 significant InDels on the host genome that were associated with susceptibility/resistance to pullorum disease. A total of 77 genes were annotated, among which MYH7, ATP2A3, and CACNA1S exhibited variations in the exons. After infection with S. pullorum, the diversity and community structure of the gut microbiota in the chicken also underwent significant changes. Lactobacillus, Escherichia_Shigella, and Klebsiella were dominant bacteria in the dead negative offspring chicks with significantly higher abundance compared to the survival negative offspring chicks. These significant changes in host genome and bacterial abundance suggest that they may be associated with the susceptibility/resistance of pullorum disease.IMPORTANCEPullorum disease can be transmitted vertically and horizontally. Population purification and antibiotic treatment are the main methods for preventing and treating this disease, but they are associated with issues, such as high cost, poor accuracy, bacterial resistance, and overused antibiotics. In traditional perspectives, research on pullorum disease primarily focused on clinical symptoms, epidemiological characteristics, and the pathogenic sites. This study, however, approaches the subject from the standpoint of host genetic basis and gut microbiota. Using the genome-wide association analysis and microbiome comparison analysis, with chicken death and survival following Salmonella pullorum infection as phenotypes, we identified significant genetic variations (e.g., MYH7, ATP2A3, and CACNA1S) and gut microbiota (e.g., Lactobacillus, Escherichia_Shigella, Bacillus, and Enterococcus_cecorum) that may relate to susceptibility/resistance of pullorum disease. These results indicate that the infection of chickens with S. pullorum and the achievement of vertical transmission may be related to the host genome and gut microbiota.