In recent years, a widespread canker disease that infects the branches of pear trees has been observed in many provinces in China; it kills the branches and results in high losses in fruit production. Symptomatic branches were collected for etiological isolation from 11 varieties of three pear species and from Malus pumila. Samples were collected from six provinces in China. In total, 143 Phomopsis isolates were obtained from 181 samples and these were identified as belonging to five species: Phomopsis fukushii (n = 69 isolates), Diaporthe eres (n = 31), P. amygdali (n = 22), P. longicolla (n = 13), and D. neotheicola (n = 8). Pathogenicity tests showed that only the first three species induced lesions on nonwounded branches of Pyrus pyrifolia var. Cuiguan. All the fungal species induced branch cankers following wound inoculations, and tests with additional pear varieties showed significantly higher virulence levels for the first three species than the latter two. A host range evaluation suggested that the five species could infect most fruit trees belonging to the Rosaceae family as well as some non-Rosaceous species. Virulence varied depending on the species of both host and pathogen. Isolates of Phomopsis amygdali had significantly higher virulence in all the tested Rosaceae plants. Correlations among the host, pathogen, and sampling regions were noted, and the morphology, growth rate, and sporulation of these species in varied media were also characterized. This study presents the first attempt to perform a broad survey and characterization of the Phomopsis spp. associated with the pear shoot cankers in China. This study shows that D. eres and P. amygdali are just as responsible for the pear shoot canker diseases as P. fukushii, and it expands the host and geographic ranges of the five species. This report provides useful information for understanding and improving management strategies for controlling this economically important disease.