Abstract:Thousands of bacteria are colonized in the intestinal tract of animals. Lactobacillus reuteri is a probiotic in animal gut and model strain for studying the evolutionary relationship between gut microbiota and host. [Objective] To study genetic diversity and host specificity of different isolates of L. reuteri, we analyzed 132 strains of L. reuteri including 116 strains isolated from human, poultry, rodents and pigs downloaded from NCBI database and 16 strains isolated from cow, sheep and horse in Inner Mongolia. [Methods] Seven housekeeping genes including ddl, pkt, leuS, gyrB, dltA, rpoA, and recA were used as targets in MLST (multilocus sequence typing) technology to study the genetic diversity of 132 L. reuteri and evolutionary relationship between strains and host habitats. [Results] All 132 strains of L. reuteri were assigned into 63 STs and 6 clonal complexes. Recombination analysis revealed that individual recombination events occurred during the evolution of L. reuteri. The eBURST and MSTree analysis showed that L. reuteri from different sources experienced distinct evolutionary processes. The 132 L. reuteri isolates were classified into 5 clusters by phylogenetic analysis, according to their correlation with the sources. [Conclusion] L. reuteri from different sources had high host specificity, indicating that L. reuteri may experience different evolutionary processes to adapt the distinct living environments.