In March 2023 dieback of shoots and branches, and cankers were observed in trees of cacao (Theobroma cacao) in plantations in four municipalities of the department of Arauca (Arauquita, Fortul, Saravena and Tame), Colombia. In Arauca, the main cacao genotypes grown are FEAR 5, FTA 2, FSA 13, and to a lesser extent the universal clone CCN 51. The incidence of diseased trees was greater in monocultures (75-85%) than in those grown under agroforestry systems (20-35%) for all genotypes (Figure 1a–b). Twenty samples, representing the four cacao genotypes (FEAR 5, FTA 2, FSA 13 and CCN 51) in the four municipalities, were taken from branches with dieback symptoms (Figure 1c–d. For each sample, plant tissue was excised from at least four lesions on the sampled branches. Two methods were used to isolate the causal pathogen. In the first, the plant tissue was disinfected and incubated in a humid chamber at 28°C in the dark for 25 days. Pycnidia were observed and were removed and cultured on potato dextrose agar (Figure 1e). For the second method the plant tissue was placed directly on water agar at 28°C for one week. A grey colony with aerial mycelium was isolated consistently from the samples (Figure 2a). The isolate was inoculated on 2% water agar with sterilised pine needles to induce pycnidia production (Crous et al., 2006). Using both isolation methods, the same pathogen was isolated. The pycnidia were crushed to release the conidia and examined under the microscope. Immature hyaline, aseptate conidia and mature brown-coloured, one-septate conidia, together with aerial grey mycelia identified the isolates as Lasiodiplodia theobromae (Figure 2). The identity of the pathogen was confirmed by PCR and DNA sequencing, and phylogenetic analyses of multiple genes. DNA was extracted from seven representative isolates, selected from the four municipalities, using a Quick-DNA™ Fungal/Bacterial MiniPrep kit (Zymo Research Group, USA). The internal transcribed spacer, and beta-tubulin and elongation factor genes were amplified by PCR using the ITS4/ITS5 (White et al., 1990), BT1a/b (Glass & Donaldson 1995) and EF (Alhudaib et al., 2022) primer pairs, respectively. Amplicons of the expected size were sequenced and sequences of representative isolates submitted to GenBank (Accession Nos. PP47166, PP481743-44, PP471668-69, PP495243-55). Phylogenetic analysis was done using MEGA version 11 (Kumar et al., 2016), concatenating the ITS and beta-tubulin and elongation factor gene sequences. All seven isolates were identified as L. theobromae (Figure 3). Pathogenicity tests were done with four isolates using detached twigs of four cacao genotypes (CCN 51, FEAR 5, FTA 2 and SA 13). The experiment was replicated three times, using five twigs per genotype in each replicate. Each twig was disinfected with 70% ethanol and 0.35% sodium hypochlorite, and a longitudinal cut was made to remove the bark. Inoculation was done by placing a 3 mm diameter mycelial plug on the wound which was covered with plastic film (Dong et al., 2020). Uninoculated agar plugs were placed on cacao twigs of the four genotypes as a control. The twigs were incubated in moist chambers at 28 ±2°C. Necrotic lesions were observed two days after inoculation; no lesions developed in the controls (Figure 4). Additionally, ten six-month-old cacao seedlings (ICS 95 genotype, the universal rootstock) were inoculated on the stem with a 3 mm mycelial plug of an actively growing colony (Úrbez-Torres et al., 2008). Seedlings showed necrosis on the petioles forming a 45° angle, stem necrosis, necrotic veins on leaflets and leaf death two weeks after inoculation (Figure 5). No symptoms developed in plants of the same age inoculated with a 3 mm plug of potato dextrose agar as a control. Lasiodiplodia theobromae has been reported as a pathogen of cacao in Cameroon, India, Malaysia and the Philippines. In Colombia, Amortegui Novoa & Becerra Betancourt (2019) suggested that it was a potential pathogen of cacao but did not provide evidence of its identification, characterisation or pathogenicity. This study represents the first report of L. theobromae causing dieback on cacao trees in Colombia. This research has been supported financially by the Sistema General de Regalías through the project "Implementación de estrategias agroforestales y vinculación de avances en el manejo agronómico y poscosecha de nuevos clones, para mejorar la productividad y calidad del cacao en el departamento de Arauca" (grant number BPIN: 2018000100148). We thank the Corporación Colombiana de Investigacion Agropecuaria for providing the infrastructure and support for the development of this research.