Dengue virus (DENV) is a medically significant flavivirus causing up to 390 million infections annually within tropical and subtropical regions. DENV infection may manifest asymptomatically, however symptomatic infections range from a relatively mild febrile disease to severe and potentially fatal febrile illness with vascular leakage. Understanding the mechanisms that underlie the virus lifecycle is paramount for the development of antiviral therapeutics and vaccines, of which none are currently available for DENV. Following cell binding and endocytosis, DENV must escape from the endosome to enable lifecycle progression. This critical process is mediated by pH-dependant structural rearrangements of the virion surface proteins precursor membrane (prM) and envelope (E). To gain further insight into the molecular mechanisms that drive DENV membrane fusion we examined the fusogenic phenotype of two prototypical laboratory strains of DENV-2, NGC and PR159, using both microscopy and biosensor based fusion assays. When compared to PR159, NGC demonstrated an enhanced fusogenic phenotype characterised by an earlier initiation of cell-to-cell fusion after infection and a higher pH threshold for fusion induction. Sequence analysis revealed a non-conservative substitution at E residue 126, where the positively charged lysine in NGC was a negatively charged glutamic acid in PR159. We propose that the glutamic acid present in PR159 could form a salt bridge with lysine at E residue 58, potentially conferring additional stability to the pre-fusion structure, explaining the phenotypic differences observed between the two strains. Functional analysis of mutant fusion proteins, in the context of a newly developed PR159 infectious clone, has the potential to validate our hypothesis and contribute to greater understanding of virus-mediated fusion, potentially providing new avenues for antiviral and/or vaccine development.