Basal stem rot incidence caused by a white-rot fungus,Ganoderma boninense, is the major disease of oil palm in Southeast Asia. The rate of disease transmission and host damage are affected by variations in pathogen aggressiveness. Several other studies have used the disease severity index (DSI) to determineG. boninenseaggressiveness levels while verifying disease using a culture-based method, which might not provide accurate results or be feasible in all cases. To differentiateG. boninenseaggressiveness, we employed the DSI and vegetative growth measurement of infected oil palm seedlings. Disease confirmation was performed through scanning electron microscopy and molecular identification of fungal DNA from both infected tissue and fungi isolated fromGanodermaselective medium. Two-month-old oil palm seedlings were artificially inoculated withG. boninenseisolates (2, 4A, 5A, 5B, and 7A) sampled from Miri (Lambir) and Mukah (Sungai Meris and Sungai Liuk), Sarawak. The isolates were categorized into three groups: highly aggressive (4A and 5B), moderately aggressive (5A and 7A), and less aggressive (2). Isolate 5B was identified as the most aggressive, and it was the only one to result in seedling mortality. Out of the five vegetative growth parameters measured, only the bole size between treatments was not affected. The integration of both conventional and molecular approaches in disease confirmation allows for precise detection.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12275-023-00040-w.