The live attenuated yellow fever (YF) vaccine was developed in the 1930s. Currently, the 17D and 17DD attenuated substrains are used for vaccine production. The 17D strain is used for vaccine production by several countries, while the 17DD strain is used exclusively in Brazil. The cell passages carried out through the seed-lot system of vaccine production influence the presence of quasispecies causing changes in the stability and immunogenicity of attenuated genotypes by increasing attenuation or virulence. Using next-generation sequencing, we carried out genomic characterization and genetic diversity analysis between vaccine lots of the Brazilian YF vaccine, produced by BioManguinhos–Fiocruz, and used during 11 years of vaccination in Brazil. We present 20 assembled and annotated genomes from the Brazilian 17DD vaccine strain, eight single nucleotide polymorphisms and the quasispecies spectrum reconstruction for the 17DD vaccine, through a pipeline here introduced. The V2IDA pipeline provided a relationship between low genetic diversity, maintained through the seed lot system, and the confirmation of genetic stability of lots of the Brazilian vaccine against YF. Our study sets precedents for use of V2IDA in genetic diversity analysis and in silico stability investigation of attenuated viral vaccines, facilitating genetic surveillance during the vaccine production process.