The French neurotropic vaccine, or FNV, was used extensively in Africa to control yellow fever (YF). Although efficacious, the vaccine caused an unacceptable rate of post-vaccinal complications in children and was subsequently replaced by the 17D vaccine. Here we report that the genomes of the wild-type YF virus French viscerotropic virus and its attenuated vaccine derivative, FNV virus from the Institut Pasteur, Paris, (FNV-IP) differ by 77 nucleotides encoding 35 amino acid substitutions. Comparison of FNV-IP and three other isolates of FNV with other YF vaccine strains (17D-204 and 17DD derived from wild-type strain Asibi) revealed that during the two attenuation processes two common nucleotide changes arose that encode two amino acid substitutions: one is in the membrane protein at amino acid 35 (M-35), the other in non-structural (NS) protein 4B at NS4B-95. These common substitutions may be important in the process of attenuation of viscerotropic disease for humans and monkeys, and/or may be involved in loss of mosquito competence of the vaccine viruses.