Lasiodiplodia, a genus in the Botryosphaeriaceae, consists of important fungal pathogens that cause leaf spots, cankers, dieback, or fruit and root rot on a wide range of hosts from tropical and subtropical regions. Species of Lasiodiplodia were isolated from asymptomatic fruit during a field survey for citrus black spot in Marion, St. Lucie, and Collier Counties, FL. Morphological characterization and single- and multi-locus phylogenetic analyses of the ribosomal DNA internal transcribed spacer region (ITS), translation elongation factor 1-alpha gene (TEF-1), and ?-tubulin gene sequences were used to identify two species, L. iraniensis and L. theobromae. Their pathogenicity was determined by inoculation of detached Valencia sweet orange or grapefruit twigs. Significant variation (P<0.0001) in length of necrotic lesions and/or cortex discoloration was detected among isolates of L. iraniensis and an interaction in terms of pathogenicity to Valencia and grapefruit among the isolates (P<0.0001). In addition, L. theobromae showed much slower development in host tissue compared to L. iraniensis. Two-year old Valencia were inoculated with mycelial plugs on pruned branches in the greenhouse. Both Lasiodiplodia spp. caused plant death (up to 2 of 5 inoculated/isolate) within 12 weeks post inoculation. Further collection from fruit and wood is underway to confirm results and the greenhouse trial will be repeated. This is the first report of L. iraniensis in the USA.