Hevea brasiliensis latex contains a large quantity of high molecular weight rubber and is thus the primary commercial source of natural rubber. Rubber and other non-rubber isoprenoids in Hevea latex are synthesised from isopentenyl diphosphate (IPP) generated from the cytoplasmic mevalonate (MVA) pathway and the plastidic methyl erythritol phosphate pathway (MEP). This study utilised two rubber tree clones (RRIM600 and PB235) that show visibly contrasting levels of yellow carotenoids for the measurement of latex isoprenoids (carotenoids, rubber and isoprenoid intermediates) and transcript levels of the genes involved in isoprenoid biosynthesis. Metabolite extraction and analysis showed that four major carotenoids namely lutein, zeaxanthin, α-carotene and β-carotene were consistently present in both RRIM600 and PB235 latex. β-carotene was found to be the major carotenoid, at 1.2 μg/g in PB235 and 0.8 μg/g in RRIM600 fresh latex samples. However, the analytical method developed to measure isoprenoid intermediates needed to be further optimised to increase extraction efficiency. To enable accurate measurement of transcript levels of key genes involved in the isoprenoid biosynthetic pathway, a set of reference transcripts was constructed by merging short-reads (RNA-seq) and long-reads (Iso-seq and full-length cDNA sequences) data from Hevea brasiliensis. This produced a comprehensive set of 193,997 transcript sequences with good level of coverage of predicted transcripts and highly conserved core plant genes. Not only did the reference transcriptome update the annotation of rubber gene models, additional transcript variants were also discovered. Manual curation of gene models for key steps associated with rubber and carotenoids resulted in a repertoire of 115 genes, with 151 corresponding transcript variants. Subsequently, differential expression analysis on the basis of mapping RRIM600 and PB235 RNA-seq reads to the reference transcriptome revealed isoform-specific expression of genes for biosynthesis of carotenoids (PSY isoform 2), IPP (AACT2 and HMGR1) and rubber (REFSRPP gene members). In addition, the levels of these genes correlated positively with the carotenoid and rubber content measurements from the same latex of PB235 and RRIM600 used for metabolite extraction. Finally, the utility of the reference transcript catalogue was demonstrated by the characterisation of the REFSRPP gene family, which is involved in rubber elongation steps. REFSRPP gene family showed a local expansion which appear to be unique to Hevea. A pilot study has demonstrated there is considerable diversity of the genomic region containing the duplicated REFSRPP genes.