Lactobacillus reuteri ( L. reuteri ) WHH1689, which was isolated from Chinese traditional highland barley wine, exhibited high survival period at room temperature in drinkable probiotic yogurt. This article aimed to indicate the genes involved in probiotic function of WHH1689 and reveal potential stress resistance based on genomic analysis. Analysis of comparative genome with closely related L. reuteri strains identified special stress adaptation. MUMmer and ACT softwares were applied for collinear analysis, and OrthoMCL program was used for sequence alignment involved in distribution of protein cluster. We identified genes coding for carbohydrate transport and enzymes, carbon metabolism pathway, gastrointestinal tract resistance, adhesive ability, and folic acid biosynthesis, etc. Genome sequence and comparative genome analysis of L. reuteri WHH1689 demonstrated specific genes for genetic adaptation and stress resistance. Tolerance, adhesion, and folate test indicated the strain had multiple probiotics. L. reuteri WHH1689 has the potential to be a probiotic candidate in dairy foods.