Technical Abstract: Fruit rot is a major pre- and post-harvest disease problem in rambutan orchards. In 2011, fruit rot was observed at the USDA-TARS orchards in Mayaguez, Puerto Rico. Infected fruit were collected and tissue sections (1 mm2) were superficially sterilized with 70% ethanol and 0.5% sodium hypochlorite. Infected fruit sections were rinsed with sterile, deionized, double-distilled water and transferred to acidified potato dextrose agar (APDA). Plates were incubated at 25ºC ± 1°C for six days. Three isolates of Calonectria hongkongensis (Cah) (CBS134083, CBS134084 and CBS134085) were identified morphologically using taxonomic keys. In APDA, colonies of Cah produced raw sienna to rust-colored aerial mycelial growth. Conidiophores of Cah had a penicillate arrangement of primary to quaternary branches of two to six phialides. Conidia (n=50) were cylindrical, hyaline, 1-septate and rounded at both ends. Conidial size was 44 to 52 µm long by 3.5 to 4.5 µm wide. Conidiophores produced terminal and lateral stipe extensions with terminal sphaeropedunculate vesicles that measured 8 to12 µm in diameter. Perithecia measured 300 to 500 µm long and 300 to 350 µm wide, were produced in groups of three, were orange to red-brown in color, and subglobose to ovoid. Asci were clavate and contained eight ascospores aggregated at the top of the ascus. Ascospores were hyaline, guttulate, fusoid with rounded ends, straight to curved, 1-septate with constriction at the septum, and measuring 28 to 36 µm long by 4 to7 µm wide. Pathogenicity tests were conducted on healthy superficially sterilized fruits. Wounded and unwounded fruit tissues were inoculated with 5-mm mycelial disks from 8-day-old pure cultures grown in APDA. Untreated controls were inoculated with APDA disks only. Fruits were kept in a humid chamber for eight days at 25°C under 12 hours of fluorescent light. Test was repeated once. Three days after inoculation (DAI), white mycelial growth was observed on fruits. Five DAI, fruits changed in color from red to brown, and yellowish mycelia colonized 50 to 62% of the surface. Eight DAI, all the fruit turned brown, mycelium growth covered the entire fruit, and conidiophores were produced on spinterns (hair-like appendages). Fruit and aril rot, and light brown discoloration were observed inside the fruit. Cah was re-isolated from diseased tissue, fulfilling Koch's postulates. Molecular identification using the ITS region of the rDNA, fragments of ß-tubulin, histone, and elongation factor (EF1-a) genes was conducted. DNA sequences of Cah were submitted to the NCBI GenBank. Accessions Nos. were: KC342208, KC342206 and KC342207 for ITS region; KC342217, KC342215, and KC342216 for ß-tubulin; KC342211, KC342209, and KC342210 for histone; and KC342214, KC342212, and KC342213 for EF1a. All gene sequences were >99% to 100% identical to ex-type specimen Cah (CBS 114828). Calonectria spp. (asexual stage: Cylindrocladium) have been associated with lychee decline syndrome in North Vietnam. Both fruits belong to the Sapindaceae family. To our knowledge, this is the first report of Cah causing fruit rot of rambutan.