Microbiota modulates plant health and appears as a promising lever to develop innovative, sustainable and eco-friendly agro-ecosystems. Key patterns of microbiota assemblages in plants have been revealed by an extensive number of studies based on taxonomic profiling by metabarcoding. However, understanding the functionality of microbiota is still in its infancy and relies on reductionist approaches primarily based on the establishment of representative microbial collections. In Arabidopsis thaliana , most of these microbial collections include one strain per OTU isolated from a limited number of habitats, thereby neglecting the ecological potential of genetic diversity within microbial species. With this study, we aimed at estimating the extent of genetic variation between strains within the most abundant and prevalent leaf-associated non-pathogenic bacterial species in A. thaliana located south-west of France. By combining a culture-based collection approach consisting of the isolation of more than 7,000 bacterial colonies with an informative-driven approach, we isolated 35 pure strains from eight non-pathogenic bacterial species. We detected significant intra-specific genetic variation at the genomic level and for growth rate in synthetic media. In addition, significant host genetic variation was detected in response to most bacterial strains in in vitro conditions, albeit dependent on the developmental stage at which plants were inoculated, with the presence of both negative and positive responses on plant growth. Our study provides new genetic and genomic resources for a better understanding of the plant-microbe ecological interactions at the microbiota level. We also highlight the need of considering genetic variation in both non-pathogenic bacterial species and A. thaliana to decipher the genetic and molecular mechanisms involved in the ecologically relevant dialog between hosts and leaf microbiota.