Chile is the main exporter of blueberries in the Southern Hemisphere, producing this fruit from October to April each year, when there is no production available in the main markets situated in the northern hemisphere. In Chile, approximately 15,600 ha is planted with highbush blueberries throughout the country. Blueberries are affected by crown and root rot diseases, which reduce productivity (Larach et al. 2009). In 2014, blueberry (Vaccinium corymbosum L. ‘Jolene’) plants showed unusual, severe wilt symptoms (incidence ranging from 50 to 90%), including leaf burn around the margins and brown discoloration of plant crowns, in two orchards of Araucanía Region (38°54′00″S, 72°40′00″W) in southern Chile. A fungal species was consistently isolated by cutting small pieces of symptomatic crowns, sterilizing with 1% sodium hypochlorite (1 min), and placing them on potato dextrose agar (PDA). Plates were incubated at 25°C for 7 days. To obtain pure fungal cultures, hyphal tips were transferred to PDA. Upon isolation, colonies formed on PDA developed hyaline aerial mycelium with hints of purple after 7 days. The fungus formed abundant macroconidia and microconidia. The macroconidia were hyaline, slightly sickle-shaped with a foot-shaped basal cell, three to five septations, and 30.34 to 50.67 μm long by 3.22 to 4.58 μm wide. Microconidia were hyaline, oval-ellipsoid to cylindrical, single-celled, and 5.47 to 13.07 μm long by 2.25 to 3.72 μm wide (n = 50). Based on cultural and morphological characteristics, the microorganism was identified as Fusarium oxysporum Schltdl (Leslie and Summerell 2006). For confirmation, DNA was extracted from four isolates, and the ITS region, 18s small subunit rRNA (18s ssu rRNA), and translation elongation factor 1-alpha (tef1-α) genes were amplified with primers ITS1/ITS4, NS1/NS4, and EF1/EF2, respectively, and sequenced. BLAST analysis of the amplified fragments showed that the four isolates were identified as F. oxysporum showing similarity with sequences for ITS (100% identity with accession no. KU986798.1 affecting V. corymbosum; isolates GenBank accession nos. MK356956, MK356957, MK356958, and MK356959), 18s ssu rRNA (99.4% identity with accession no. KP027007.1 affecting Acacia hindsii; isolates GenBank accession nos. MK355436, MK355437, MK355438, and MK35439), and tef1-α (99% identity with accession no. KM092478.1 affecting Vaccinium sp. and 99.4% identity with accession no. MG356947.1 affecting Passiflora edulis; isolates GenBank accession nos. MK414771, MK414772, MK414773, and MK414774) available in the GenBank for this pathogen. To test Koch’s postulates, the crown section of blueberry plants cultivar Jolene (n = 10) was inoculated with 200 µl of 1 × 10⁶ conidia/ml suspension on a 10-mm wound made with a scalpel on the crown base. Inoculated plants were placed in a greenhouse at room temperature (20 to 25°C) for 30 days. After 2 weeks, all inoculated plants produced the same symptoms observed in the field. No symptoms were observed on the negative control plants, which only were inoculated with 200 µl of sterile distillate water (n = 2). Small pieces (3 × 3 mm) of tissue from lesion margins were cultured on PDA. Fungi emerging from tissue pieces were identified based on morphology as F. oxysporum. The experiment was conducted once with the four genetically identified isolates (two replicates per isolate). Although Fusarium wilt of blueberry is reported in China (Liu et al. 2014), to our knowledge, this is the first report of Fusarium wilt on blueberry caused by F. oxysporum in Chile, and this pathogen should be considered among the possible crown and root rot pathogens affecting this important berry crop in the country.