Targeting regions of proteins that show a high degree of structural conservation has been proposed as a method of developing immunotherapies and vaccines that may bypass the wide genetic variability of RNA viruses. Despite several attempts, a vaccine that protects evenly against the four circulating Dengue virus (DV) serotypes remains elusive. To find critical conserved amino acids in dengue viruses, 120 complete genomes of each serotype were selected at random and used to calculate conservation scores for nucleotide and amino acid sequences. The identified peptide sequences were analysed for their structural conservation and localisation using crystallographic data. The longest, surface exposed, highly conserved peptide of Envelope protein was found to correspond to amino acid residues 250 to 270. Mutation of this peptide in DV1 was lethal, since no replication of the mutant virus was detected in human cells. Antibodies against this peptide were detected in DV naturally infected patients indicating its potential antigenicity. Hence, this study has identified a highly conserved, critical peptide in DV that is a target of antibodies in infected humans.