Diaporthe ampelina, causal agent of Phomopsis cane and leaf spot of grapevine (Vitis viniferaL.) is isolated frequently from grapevine wood cankers, causing Phomopsis dieback. The latter disease is associated with four other Diaporthespecies, three of which also are reported from hosts other than grape. To better understand the role of this Diaporthecommunity in Phomopsis dieback of grapevine and the potential for infection routes among alternate hosts, 76 Diaportheisolates were recovered from wood cankers of cultivated grape, pear, apricot, almond and the wild host willow in four California counties. Isolates were characterized morphologically and assigned to species based on multigene sequence analyses. This study identified eight Diaporthespecies from grapevine and one novel taxon from willow, D. benedicti. We report the first findings of D. australafricanaand D. novemin North America. Our findings also expand the host ranges of D. ambiguato apricot and willow, D. australafricanato almond and willow, D. chamaeropisto grapevine and willow, D. foeniculinato willow and D. novemto almond. The generalists D. ambiguaand D. ereswere the most genetically diverse species, based on high nucleotide and haplotypic diversity, followed by the grapevine specialist D. ampelina. Analyses based on multilocus linkage disequilibrium could not reject the hypothesis of random mating for D. ambigua, which is further supported by relatively high haplotypic diversity, reports of both mating types and reports of successful matings in vitro. Pathogenicity assays revealed that D. ampelinawas the most pathogenic species to grapevine wood.