Vanilla is an economically important crop for low‐lying humid tropical regions, but cultivated plants face serious phytosanitary problems. Fusarium wilt is a devastating disease affecting vanilla crops, caused by the fungal pathogens Fusarium oxysporum f. sp. vanillae (Fov) and F. oxysporum f. sp. radicis‐vanillae (Forv), part of the F. oxysporum species complex (FOSC). We characterized 29 fungal isolates from a vanilla crop and crop wild relatives (CWR) using molecular (EF1‐α and ITS‐rRNA loci) and morphological traits. Fusarium was the predominant genus, followed by Colletotrichum and Clonostachys. Four Fusarium species were identified: F. oxysporum (37.9%), Fusarium solani (20.7%), Fusarium pseudocircinatum (13.8%) and Fusarium concentricum (10.3%). The latter three species were isolated only from CWR and may represent latent pathogens. Fov was isolated from both the crop and CWR, while a Forv‐affiliated isolate was also found in a vanilla crop, marking the first report in the neotropical region. The EF1‐α locus provided greater genotype resolution, as well as having reference sequences for Forv. However, the fungal barcode ITS locus is widely applied. We recommend the continued use of both loci for Fusarium diagnosis in vanilla to facilitate early detection and the development of effective integrated crop management strategies.