The bile salt hydrolase (BSH) activity is responsible for the cholesterol-lowering effect of the probiotic strains. The present study aimed to investigate the relationship between bsh gene-expression (GE) levels responsible for the BSH activity and the parameters of bile salt resistance of different Lactobacillaceae species. Accordingly, 11 Lactobacillaceae family strains with high cholesterol assimilation ratio (49.21–68.22%) determined by the o-phthalaldehyde method selected from 46 Lactobacillaceae species was evaluated for their features including acid tolerance, bile tolerance, and BSH activity. All tested strains survived at pH 2 medium and 0.3% (w/v) bile salt and showed positive BSH activity for glycocholic acid (GCA) and taurocholic acid (TCA). BSH gene expression was performed to provide clear information and to identify the key genes responsible for BSH activity. bsh3 genes were found highest GE level (P < 0.05) in Lactiplantibacillus plantarum and Lacticaseibacillus paracasei strains. The results showed that high cholesterol assimilation ratio were closely correlated with BSH activity and the parameters of bile salt resistance. The results of this study will support the development of a new approach based on phenotypic and genetic analysis to determine the bile salt parameters. The study will be useful for the selection of Lactobacillus strains with high bile salt resistance.