Diplodia sapinea is a cosmopolitan endophyte and opportunistic pathogen occurring on several conifer species in Europe for at least 200 years. In Europe, disease outbreaks have increased on several Pinus spp. in the last few decades. In this study, the genetic structure of the European D. sapinea population was investigated using thirteen microsatellite markers. In total, 425 isolates from 15 countries were analysed. A high clonal fraction and low genetic distance between most populations was found. One single haplotype dominates the European population, being represented by 44% of all isolates and found in nearly all investigated countries. Three genetically distinct subpopulations were found: Central/North European, Italian and Georgian. The recently detected populations of D. sapinea in northern Europe (Latvia, Estonia and Finland) share several haplotypes with the German population, suggesting introduction from Central Europe. The northern European populations show similar genetic diversity to those in Central Europe suggesting either that the fungus has existed in the North in an asymptomatic mode for a long time or that it has spread recently by multiple introductions. Although this fungus reproduces predominantly asexually, considerable genetic diversity was found even among isolates of a single tree. According to currently published allelic patterns, D. sapinea most likely originates from North America. In order to enable the detection of endophytic or latent infections of planting stock by D. sapinea, new species-specific PCR primers were designed. During the search for Diplodia isolates, we identified D. africana in California, USA, which is the first record of this species in North America.