Members of the Botryosphaeriaceae family are known to cause Bot gummosis on many woody plants worldwide. To identify pathogens associated with Bot gummosis on citrus in California, scion and rootstock samples were collected in 2010 and 2011 from five citrus-growing counties in California. Symptoms observed on citrus included branch cankers, dieback, and gumming. Various fungal species were recovered from necrotic tissues of branch canker and rootstock samples. Species were identified morphologically and by phylogenetic comparison as ‘Eureka’ lemon, ‘Valencia’, ‘Washington Navel’, ‘Fukumoto’, grapefruit, ‘Satsuma’, and ‘Meyer’ lemon. Species were identified morphologically and by phylogenetic comparison of the complete sequence of the internal transcribed spacer regions, β-tubulin gene, and elongation factor α-1 genes with those of other species in GenBank. A consensus-unrooted most parsimonious tree resulting from multigene phylogenetic analysis showed the existence of three major clades in the Botryosphaeriaceae family. In total, 74 isolates were identified belonging to the Botryosphaeriaceae family, with Neofusicoccum spp., Dothiorella spp., Diplodia spp., (teleomorph Botryosphaeria), Lasiodiplodia spp., and Neoscytalidium dimidiatum (teleomorphs unknown) accounting for 39, 25, 23, 10, and 3% of the total, respectively. On inoculated Eureka lemon shoots, lesion length was significantly different (P < 0.05) among 14 isolates recovered from portions of cankered tissues of the original trees. Lesion lengths were significantly longer (P < 0.05) for shoots inoculated with isolates of Neofusicoccum luteum and shorter for shoots inoculated with isolates of Dothiorella viticola (P < 0.05) than those of other species. Identifying the distribution and occurrence of these fungal pathogens associated with Bot gummosis is useful for management applications during occasional outbreaks in California.