Aims: To investigate the changes in bacterial diversity on fresh spinach phyllosphere associated with storage at refrigeration temperatures. Methods and Results: Community structure and population dynamics of spinach phylloepiphytic bacteria associated with packaging and refrigeration of ready-to-eat fresh produce were evaluated using pyrosequencing of 16S rRNA gene amplicons. A diverse community responsive to storage at refrigerated temperatures was detected belonging to over 1000 operational taxonomic units, including many diverse members not previously described on the phyllosphere. Of the approx. 8800 unique sequences examined from fresh spinach leaves, 75% were from previously undescribed taxa. The classified sequences from the fresh spinach phyllosphere were assigned to 11 different phyla with the largest number of reads belonging to Proteobacteria and Firmicutes. Packaging and storage of spinach under refrigerated conditions decreased the richness, diversity and evenness of the bacterial community. Refrigeration at 4 and 10 degrees C and storage resulted in a decrease in number of taxa represented from 11 phyla in fresh spinach to only 5 phyla after 1 day of storage. Sequences belonging to gamma-Proteobacteria, particularly Pseudomonas spp. and members of the Enterobacteriaceae, were the most numerous after 15 days of storage at both temperatures. Growth inhibition of the genera Escherichia was achieved at 4 degrees C but not at 10 degrees C storage, thus highlighting the importance of temperature in fresh packaged spinach. Conclusions: The application of pyrosequencing to describe composition and diversity of the phyllosphere on spinach leaves provided a broader outlook of the bacterial composition of this community complementing other phyllosphere studies that have used culture- and nonculture-dependent approaches. Significance and Impact of the Study: Pyrosequencing allowed a broader description of the bacterial composition and diversity of the spinach leaf surface than previously obtained using culture-based detection and will be a powerful tool to help ensure the future safety and quality of packaged spinach.