A maximum-likelihood approach was used to analyse selection pressures acting on genes from all four serotypes of dengue virus (DEN). A number of amino acid positions were identified within the envelope (E) glycoprotein that have been subject to relatively weak positive selection in both DEN-3 and DEN-4, as well as in two of the five genotypes of DEN-2. No positive selection was detected in DEN-1. In accordance with the function of the E protein as the major antigenic determinant of DEN, the majority of these sites were located in, or near to, potential T- or B-cell epitopes. A smaller number of selected sites was located in other well-defined functional domains of the E protein, suggesting that cell tropism and virus-mediated membrane fusion may also confer fitness advantages to DEN in nature. Several positively selected amino acid substitutions were also identified in the NS2B and NS5 genes of DEN-2, although the cause of this selection is unclear, whereas the capsid, membrane and non-structural genes NS1, NS2A, NS3 and NS4 were all subject to strong functional constraints. Hence, evidence was found for localized adaptive evolution in natural isolates of DEN, revealing that selection pressures differ among serotypes, genotypes and viral proteins.