Low temperature is a primary factor limiting geographical distribution of plants and crop yield in large areas of the world. Plant-associated microbial communities have rarely been acknowledged as possible determinants of cold acclimation, the process leading to freezing tolerance in plants. Here we studied the impact of cold acclimation on leaf-associated bacteria by analyzing 10 different Arabidopsis ecotypes differing widely in their freezing tolerance. In climate chamber experiments, we found that leaves of all Arabidopsis accessions were colonized by highly diverse bacterial taxa (852 operational taxonomic units) mainly from Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. During cold acclimation, in all ecotypes the bacterial community composition and diversity, and especially the core species composition changed drastically. However, the response of the bacterial communities was highly ecotype-dependent; sensitive and tolerant accessions shared only eight responders. Unique responders in tolerant accessions were identified as putative indicators of freezing tolerance. Thus, leaf bacteria appear to be genotype-dependent associated with cold acclimation, which suggests an additional function of plant−microbe interaction and may open new possibilities for biotechnological applications.