Peach (Prunus persica (L.) Batsch) is produced locally in Yangshan, Wuxi City, China. In recent years, a widespread shoot blight has been observed in many peach orchards of Yangshan that kills the twigs and results in high losses in fruit production. Disease incidences ranged from 10 to 20% in the affected orchards and, in extreme cases, 40% of the trees were affected. Shoot blight of peach is caused by a fungus, previously identified as Phomopsis amygdali. Between 2014 and 2015, samples were collected four times from three peach orchards located in Yangshan to understand the etiology of shoot blight. Interestingly, two types of shoot blight symptoms were observed: one characterized by necrotic lesions with rings and one without rings. Based on conidial morphology, cultural characteristics, and analysis of nucleotide sequences of three genomic regions (the internal transcribed spacer region, a partial sequence of the β-tubulin gene, and the translation elongation factor 1-α), isolates were identified as P. amygdali and Botryosphaeria dothidea. Remarkably, most of the P. amygdali isolates were recovered from twigs showing necrotic lesions without rings. In contrast, most of the B. dothidea isolates were recovered from twigs with rings in the necrotic lesions. Correlations among pathogens, sampling regions, and disease symptoms were noted, and growth rates of these pathogens were characterized. Pathogenicity tests showed that B. dothidea isolates could induce necrotic lesions with rings but P. amygdali isolates could only induce necrotic lesions. Moreover, the B. dothidea isolates exhibited higher levels of virulence than P. amygdali isolates on the peach twig. Additionally, high frequencies of detection of both P. amygdali and B. dothidea from buds indicated that buds may be the primary site of fungal invasion. Cankers and necrotic twigs may also serve as infection courts. Our results suggest that B. dothidea and P. amygdali are the common causal agents of peach shoot blight in Yangshan, China. This finding provides a basis for the development of effective management strategies.