Brevibacillus laterosporus has been used as a direct-fed microbial additive in chicken feed owing to its strain safety and the ability to produce broad-spectrum antimicrobial peptides. To assess the efficacy and persistence of Brevibacillus laterosporus, it is necessary to track the supplied strains throughout the gastrointestinal tract. This study aims to evaluate the in vitro probiotic properties of Brevibacillus laterosporus S62-9 and its capacity to colonize and persist in the intestines of broiler chickens. Results indicated that the spores of Brevibacillus laterosporus S62-9 exhibited higher tolerance to the digestive condition, and higher adhesion rate to mucus and intestinal cells compared to vegetative cells. The spores remained dormant during digestion in the crop and stomach but germinate in the small intestine with a germination rate exceeding 70%. Subsequently, the whole genome of S62-9 was sequenced, and specific primers were designed and validated based on comparative genome analysis. The quantitative PCR-based method had a detection limit ranging from 2.71 to 7.71 log10 CFU/mL in pure culture and from 3.25 to 9.25 log10 CFU/g in broiler cecal content. In vivo colonization assay indicated that the Brevibacillus laterosporus S62-9 can transiently colonize the broiler cecum. In summary, our results indicated that the Brevibacillus laterosporus S62-9 spores exhibited high tolerance to the digestive tract environment, germinated in the intestine, and establish colonization in the cecum of broilers. The findings provide a theoretical basis for the future application of Brevibacillus laterosporus S62-9 in broiler farming.