Botryosphaeria dothidea causes diseases in many different hosts worldwide. This species has become the most prominent fungal taxon causing grapevine trunk disease in China, with a recent disease outbreak. To reveal the genetic diversity and explore the origin of B. dothidea, six novel microsatellite markers were developed and used for the genotyping of 151 isolates obtained from China and overseas. The results demonstrated the high genetic diversity of the B. dothidea populations. Bayesian cluster analysis separated the total B. dothidea isolates into five genetic populations. B. dothidea isolates from Chinese grapevines were observed to share alleles with isolates from different hosts within China and from grapevines growing overseas, indicating both endemic host shifts and exotic introduction. In addition, unique pathogen genotypes were identified in Chinese grapevine isolates. Hence, we infer that B. dothidea isolates from multiple origins are contributing to the dieback and canker outbreak currently occurring in China.