Lactobacillus rhamnosus(L. rhamnosus) is a symbiotic bacteria in the intestines of humans and animals and has applications in many fields， including food， medicine and animal farming. In this paper， the whole genome of L. rhamnosus Probio-M9 was used as the object to analyze the genome differences of different Lactobacillus rhamnosus combined with the 214 L. rhamnosus genome sequences published by NCBI. The results showed that a total of 16 915 pan-genes and 247 core genes were identified in 215 strains of L. rhamnosus； the subsequent phylogenetic trees were constructed using 247 core genes found that there was no obvious clustering trendx between the source and the place of separation. L. rhamnosus Probio-M9 was located in Branch Ⅱ， which had the largest branch. The genetic relationship among strains of this branch was close， the difference was small， and the distinction was difficult. The RAST annotation and CAZy annotation analysis of 98 strains of L. rhamnosus in BranchⅡ found that L. rhamnosus had a high degree of similarity in terms of function， but some of the strains were different from L. rhamnosus Probio-M9. Compared with other L. rhamnosus， L. Rhamnosus Probio-M9 had stronger ability to regulate its own metabolism， transcription， transportation and other aspects and contains genes related to probiotics， such as glutathione (gshAB)， exopolysaccharides (RMLA-RMLD， epsH) and genes related to enhancing host metabolic capacity (tagE). The genomic differences of L. rhamnosus Probio M9 were revealed by comparative genomics， which laid the foundation for the development and application of L. rhamnosus Probio-M9.