Pitaya (Hylocereus spp.) is a cactus cultivated in regions with a tropical and subtropical climate. In Brazil, the installation of pitaya orchards has spread nationwide, and some diseases associated with phytopathogens have been reported. In Pará, a state located in the North of Brazil, the constant association of a fungus in pitayas, characteristic of the genus Neoscytalidium, was observed, manifesting symptoms of stem canker. Therefore, this study aimed to identify and confirm the causal agent associated with stem canker in pitayas (H. costaricensis) in Pará, Brazil. Symptomatic pitaya cladodes were used to isolate the pathogens. Subsequently, the macroscopic and microscopic characteristics of the colonies, molecular analysis, and pathogenicity test were carried out. The pathogen showed rapid growth on PDA, with a diameter of approximately 8–9 cm, after 4–5 days of incubation at 25 °C with light gray colonies in 5 days and dark gray in 14 days. Microscopic evaluation showed septate and branched hyphae. The conidia are ellipsoid to ovoid, with smooth walls and disarticulated, and may have one or zero septa, with an average size of 7.2–12x3-7.8 μm. Molecular analysis, using the ITS and TUB2 sequences, showed 100% identity with the phytopathogenic fungus Neoscytalidium dimidiatum, according to BLAST analysis. In the phylogenetic tree, the isolate was close to isolates from China and Vietnam. Furthermore, the pathogenicity test confirmed the fungus Neoscytalidium dimidiatum as a causal phytopathogen associated with stem canker in pitaya (Hylocereus costaricensis) in Pará, Brazil. These findings may assist in future studies to manage the disease.