Ganoderma boninense is a telluric lignicolous basidiomycete and the causal agent of basal stem rot, one of the most devastating diseases of the oil palm (Elaeis guineensis). While the fight against G. boninense is of major concern in Southeast Asia, little information is available regarding the genetic diversity and evolutionary history of the fungus. In this context, the development of an informative molecular marker set to characterize the diversity of G. boninense is a key step towards understanding the biology of this pathogen. A G. boninense draft genome sequence of 63 Mbp, assembled using 454 and Illumina sequencing technology, was used to identify and develop a set of microsatellite markers (simple sequence repeats, SSRs). A total of 2487 SSRs were identified, for which 145 SSR primer pairs were designed. These SSRs are characterized by di- to hexanucleotide motifs with 5 to 34 repetitions. Ninety-seven SSR loci were successfully amplified on an initial small set of G. boninense isolates from Indonesia. A collection of 107 isolates from several regions in Southeast Asia were screened to characterize each locus for allele number, polymorphism information criterion and the presence or absence of null alleles at each locus. These results allowed us to propose an effective set of 17 SSRs for studying genetic diversity within G. boninense.