Simple SummaryLigilactobcillus salivarius is a Gram-positive bacterium that commonly colonizes the mucous membranes of the digestive tracts of humans and animals, including birds. It belongs to the group of lactic acid bacteria which, by producing lactic acid, acidify the intestinal environment and limit the development of undesirable intestinal microflora. In addition, L. salivarius can produce other antimicrobial substances, such as bacteriocins and hydrogen peroxide. Due to limiting the development of unfavourable microflora and other health-promoting effects, L. salivarius bacteria are considered as potential probiotics that may increase animal health, and thus animal production indicators. In this work, we undertook research on the characteristics of L. salivarius strains from chickens, geese, turkeys and domestic pigeons. We showed great variation in phenotypic and genotypic traits between strains and the evolutionary adaptation of L. salivarius strains to the colonization of a specific host. The results of the study contribute to knowledge of the characteristics of the species L. salivarius and may be useful in the selection of probiotic strains.Ligilactobacillus salivarius is an important member of the human and animal gut microbiota, and selected strains are promising probiotics, but knowledge of the characteristics of avian isolates is still limited. In this study, we examined selected phenotypic and genotypic traits of 33 L. salivarius strains from geese, chickens, turkeys and pigeons. The strains varied in terms of cell size, colony morphology, broth growth characteristics, biofilm formation, tolerance to bile, hydrophobicity and phenotypic and genotypic antibiotic resistance profiles. Large variation among strains was noted for the utilization of sorbitol, salicin, trehalose, rhamnose, inulin and N-acetyl-D-glucosamine. The presence of genes related to sugar metabolism, i.e., mipB, tktA, rhaB and LSL_1894, was not always correlated with the biochemical phenotypic profile. Correlations were recorded between the host and utilization of certain sugars as well as tolerance to bile. The repA-type megaplasmid and genes coding for Abp118 bacteriocin were detected in 94% and 51.5% of L. salivarius strains, respectively. Phylogeny based on groEL gene sequences was partly correlated with the origin of the strains and revealed an evolutionary distance between L. salivarius strains from humans and birds. The results of the study contribute to knowledge of the characteristics of the species L. salivarius. Intraspecies variations of L. salivarius strains may affect their ability to colonize specific niches and utilize nutrients and reveal potential strain-dependent effects on host health.