MPOB has initiated an oil palm genetic resources programme which includes the collection, evaluation, utilisation and conservation of oil palm germplasm. The objectives of this study were to determine the potential use of expressed sequence tag-simple sequence repeats (EST-SSR) markers to study the genetic variability of the germplasm collection. A total of 330 palms originating from 11 countries in Africa were screened using 10 EST-SSR primers. The data were analysed using the Biosys-1 software to calculate the genetic variability parameters. It was found that the germplasm exhibited a high level of genetic diversity. Most of the loci tested were 100% polymorphic at 0.95 criterion. A total of 46 alleles were detected across all the germplasm populations. Of these, three were considered as rare alleles. The Nigerian germplasm showed the highest number of alleles per locus and the highest number of rare alleles, a high percentage of polymorphic loci and high heterozygosity, suggesting that Nigeria could be the centre of diversity of the wild oil palm. This study also revealed that the Madagascar germplasm is unique and different compared with the oil palm populations from the African mainland. Based on the dendrogram constructed, the germplasm populations could be divided into three major clusters; Cluster 1 consisting of Angola, Tanzania, Cameroon, Nigeria, Democratic Republic of Congo (formerly known as Zaire), Sierra Leone, Guinea and Ghana germplasm, Cluster 2 consisting of Gambia and Senegal germplasm, while the Madagascar germplasm was placed in Cluster 3. The mean genetic distance across the MPOB germplasm populations was 0.251.