Peach bacteria leaf shot hole is a commonly occurring foliar disease of economic importance, which severely affects the host photosynthesis and contributes to substantial losses in yield each year. To date, however, the causal agents of this disease have yet to be conclusively identified. Based on morphological, physiological, and biochemical characteristics, and the results of multi-locus sequence analysis (MLSA), the causal pathogens were identified as the bacteria Pantoea agglomerans and Pantoea ananatis. To the best of our knowledge, this is the first report of the peach bacterial shot hole being caused by P. ananatis. The two pathogens can induce a clear hypersensitive response in tobacco leaves, indicating the isolates have strong pathogenic potential. Analysis of the biological characteristics of these pathogens indicated that they have good viability at temperatures ranging from 22 to 40 °C, with optimal temperatures for the growth of both species lying between 28 and 34 °C. However, P. ananatis was found to show slightly greater tolerance to high temperatures than P. agglomerans, the former being able to survive at 59 °C, whereas the latter cannot. The two species are also able to grow well over a broad range of pH from 4.0 to 9.0, with both growing optimally at pH 7.0, indicating that these pathogens are neutrophilic. In addition, they also show strong saline tolerance. These findings will contribute to gaining a more complete understanding of the factors contributing to disease epidemics and the development of effective disease control strategies.