A genetic algorithm-based RNA secondary structure prediction was combined with comparative sequence analysis to construct models of folding for the distal 380 nucleotides of the 3'-untranslated region (3'-UTR) of yellow fever virus (YFV). A number of structural elements that are thermodynamically stable, conserved in shape, and confirmed by compensatory mutations were revealed. At the same time structural polymorphisms were observed among strains of YFV. These polymorphisms showed an association with virulence: all wild and pathogenic strains were likely to be folded in a significantly different way from vaccine strains with reduced virulence. Structural divergence was also found among vaccine strains, with 17DD, the most virulent in the mouse model, exhibiting an intermediate pattern of folding, combining structural features of both wild and vaccine strains. The observation of a strong association between secondary structure of the 3'-UTR and virulence of YFV may help elucidate the molecular mechanisms of virus attenuation and lead to new strategies of vaccine development directed towards rational modification of secondary structure of the 3'-UTR.