Rubber elongation factor (REF) is a 14.6 kDa stress related family protein, which plays a major role in the chain elongation process of natural rubber biosynthesis pathway in Hevea. Due to its significance in rubber biosynthesis, molecular markers developed from REF gene will be ideal for genetic analysis and association studies related to yield and rubber quality in Hevea. Attempts were made to characterise REF gene in different genotypes to identify sequence polymorphisms like SNPs and indels. Phylogenetic analysis was performed to understand their similarity/dissimilarity with homologs in other plant species as well. Genomic region of REF gene was amplified and sequenced from five different genotypes to identify nucleotide variations among them and haplotype blocks were constructed using the Single Nucleotide Polymorphisms (SNPs) identified. A total number of 32 novel SNPs were identified with a frequency of 1 in every 53 bases. Five haplotypes were constructed from the SNP data and their impact at the functional level was estimated by gene expression studies. The SNP and generated haplotype data from the study has wide applications in different areas of Hevea crop improvement like assessment of genetic diversity, characterisation of cultivated clones and wild accessions, yield related marker development etc. Additionally results from this study further ascertain distinctive nature of REF gene in Hevea which may be partly responsible for its high rubber producing capability.