Objective: To investigate the genetic stability of Lacticaseibacillus rhamnosus Probio-M9 (L. rhamnosus Probio-M9) during continuous passage. Methods: The morphological changes， carbohydrate utilization capacity， activity and metabolic characteristics of L. rhamnosus Probio-M9 were compared in the 0， 25， 50， 75 and 100 passages of the probiotic strain. At the same time， comparative genomics was used to analyze the changes of genome level during the continuous culture of 100 generations. Results: There were no significant changes (P > 0.05) in phenotypic characteristics and metabolic characteristics of L. rhamnosus Probio-M9 during 100 generations of continuous culture， and the phenotypic characteristics were stable， and the strain activity was maintained well. At the same time， using the genome of L. rhamnosus Probio-M9 as the reference sequence， 15 strains of different generations were sequenced， and the genome size and GC content showed no significant difference between the 15 strains cultured continuously and L. rhamnosus Probio-M9. Phylogenetic relationships showed that the 15 strains were closely related to the original genome. From the identified SNP loci， no mutation locus with stable inheritance was found in every two generations， and the collinear strain was good. The mutation level of the coding gene at the genome level confirmed that L. rhamnosus Probio-M9 had stable phenotypic characteristics and metabolic characteristics during the continuous culture of 100 generations. Conclusion: L. rhamnosus Probio-M9 has good genetic stability during continuous passage， which provides a theoretical basis for further development， research and industrialization of L. rhamnosus Probio-M9.