The genetic stabilities of the three attenuation loci of the candidate dengue 2 (D2) PDK-53 vaccine virus were evaluated for the PDK-53 virus and PDK-53-vectored chimeric D2/1, D2/3, and D2/4 viruses following 10 sequential passages in Vero cells. Sequencing revealed that the dominant NS1-53-Asp and the NS3-250-Val attenuation loci were extremely stable, whereas reversion occurred at the 5'NCR-57-U locus in 10 of the 18 viral lineages tested. A more sensitive and quantitative assay, the TaqMan mismatch amplification mutation assay (TaqMAMA), was employed to more finely discriminate the level of reversion at the 5'NCR-57 locus. This rapid genetic assay permitted detection of 80% in the viral population. Chimeric viruses based on the PDK-53-V (all three mutations present) genetic background were more stable than those developed in the PDK-53-E (5'NCR and NS1 mutations present) background. The TaqMAMA can be applied in quality control analyses to ensure that attenuated vaccine seeds contain undetectable or minimal levels of reversion at a given attenuation locus.