Aims: Basal stem rot (BSR) disease caused by Ganoderma pathogenic fungi, especially Ganoderma boninense is thriving rapidly in both areas with coastal and inland soils. The objectives of this study were to isolate and characterize Ganoderma isolates collected from various locations in Peninsular Malaysia through the comparison of their growth rate in vitroly on conventional and novel palm extract media, and to determine the degree of virulence caused by the isolates in oil palm seedlings. Methodology and results: In this study, 12 Ganoderma isolates were collected from infected oil palm trees, from various locations – Johor, Negeri Sembilan, Kedah, Perak, Pahang, and Kelantan, in Malaysia in year 2011. Twelve Ganoderma isolates were identified using molecular method with primer set that targeted at small-subunit 18S rDNA fragment, and characterized by determining the in vitro growth rate, and degree of virulence in 2-month-old oil palm seedlings in the nursery using both disease incidence (DI) and disease severity index (DSI) as the measurements to quantify the infection. All the Ganoderma isolates were identified as G. boninense and sequences of the respective isolates were deposited in GenBank. In general, all the isolates proliferated faster on oil palm extract medium (OPEM) compared to malt extract agar (MEA). Twelve G. boninense isolates were observed to illustrate different degree of virulence ranging from highly pathogenic to least pathogenic. Conclusion, significance and impact of study: Cultures of 12 G. boninense isolates were observed to show faster growth rate (P < 0.014) on OPEM under in vitro conditions compared to conventional MEA medium, except Bt Lintang G10 and GBA G12 isolates. OPEM medium could provide a better alternative for maintaining and culturing Ganoderma strains. In the current study, both DI and DSI were highly correlated. However, there were low linear relationships (R2 < 0.423) between mycelia growth rate (on MEA and OPEM) and degree of virulence (DI and DSI) at 12-, 14- and 16- weeks after treatments among the G. boninense isolates tested. Furthermore, different degrees of virulence in twelve separate Ganoderma isolates were reported. Therefore, it is crucial to incorporate more than one isolate into any researches on screening for Ganoderma resistance or tolerance planting materials, searching for potential biological control agents, and studying bitrophic or tri-trophic interactions. In addition, this study was aimed to isolate G. boninense strains with various virulence levels for future studies.