Objective: In this study， the genetic stability of Bifidobacterium animalis subsp. lactis V9 with excellent prebiotic properties was used as the study object to study its genetic stability in continuous passage. Methods: When the Bifidobacterium animalis subsp. lactis V9 was continuously transmitted in TPY medium for 100 generations， the colony morphology， carbohydrate utilization and combined comparative genomics of 0， 25， 50， 75 and 100 generations were comprehensively analyzed. Results: There were no significant differences in phenotypic characteristics such as bacterial morphology and carbohydrate utilization of Bifidobacterium animalis subsp. lactis V9 during the continuous passage process (P>0.05)， And with the original genome of the Bifidobacterium animalis subsp. lactis V9 as a reference sequence， The study found that the genome size and GC content of the Bifidobacterium animalis subsp. lactis V9 at different generations were not significantly different from the original genome (P>0.05)， and the phylogenetic tree results showed that the Bifidobacterium animalis subsp. lactis V9 at different generations was closely related to the original genome， and by identifying the SNP mutation site， it was found that the genome of the Bifidobacterium animalis subsp. lactis V9 at different generations was conservative， and no stable genetic mutation sites were found， and further carbohydrate metabolism active enzyme annotation was carried out， and it was found that the strains at different generations had similar carbohydrate metabolism-related genes， and the genetic characteristics were highly similar. Conclusion: The Bifidobacterium animalis subsp. lactis V9 has excellent genetic stability during continuous passage， which provides a theoretical basis for the further development and industrialization of probiotics.