Eurycoma longifolia has been widely used for various traditional medicinal purposes in Malaysia. Among the major compounds produced in E. longifolia is quassinoids. However little is known about the biosynthesis pathway leading to these compounds. As a starting point, genes involved in terpenoids biosynthesis pathway, which is the primary pathway leading to quassinoid were analysed using Illumina high-throughput RNA-sequencing technology. The transcriptome profiles were generated from roots of the mature 10-year-old and the young 1-year-old E longifolia. BLAST against the Nr database of 60,753 non-redundant unigenes obtained indicated that only 34,673 (57%) showed homology to known proteins and was highly similar to citrus. Most assignments of gene ontology based on the biological process category were to “metabolic process”. KEGG analysis showed that key enzymes in major secondary metabolite pathways were present in the transcriptome and the highest were assigned to “phenylpropanoid biosynthesis” and “terpenoid backbone biosynthesis”. Differentially expressed gene analysis predicted 154 unigenes were potentially related to quassinoid biosynthesis. They were up-regulated in 10-year-old roots and were either involved in terpenoids backbone biosynthesis,encoded for transcription factors (WRKY and AP2/ERF genes) or encoded for cytochrome P450.