Trunk diseases such as esca have been recognized as an economically important problem of grapevine worldwide. A study was conducted to characterize the distribution of Phaeomoniella chlamydospora in Chile. A field survey of young and mature grapevines from 67 vineyards located along a 1,315-km north-south axis demonstrated that P. chlamydospora was present in 94.9% of the grapevine samples showing the black-wood streaking symptom (BWS) but not the characteristic foliar symptoms of esca. Phylogenetic analysis of the ribosomal DNA internal transcribed spacer (ITS) combined with β-tubulin (BT) genes grouped Chilean isolates together with reference isolates from South Africa and the United States, whereas Spanish isolates were clustered separately. Chilean isolates differed by only 2 to 3 bp for BT and ITS, respectively. Conidia germinated at 5 to 35°C, with an optimal temperature of 25 to 30°C. Isolates were pathogenic, and Koch's postulates were fulfilled in separate sets of inoculations of axenic plantlets, cuttings, 2-year-old plants, spurs, and shoots of V. vinifera. This study showed that P. chlamydospora was associated consistently with BWS and no other apparent symptom in young and mature grapevines, including nursery plants, in Chile. Inoculum was absent from the soil, grapevine pruning debris, sap samples, and herbaceous weeds, which is in contrast to past studies. At this time, Vitis spp. are the only known hosts of P. chlamydospora in Chile.