Over the last 17 years, the yellow fever (YF) 17DD vaccine secondary seed lot 102/84 was used to produce many million doses of vaccine but it was recently used up. In the absence of other lots at the same passage level a large vaccine batch produced from 102/84 was turned into a new working seed. This new seed was characterized with regard to attenuation in the recommended internationally accepted monkey neurovirulence test (MNVT) using the 102/84 virus as reference. All rhesus monkeys (Macaca mulatta) developed limited viremia and comparable neutralizing antibody titers. Clinical evaluation and histological examination of the central nervous system (CNS) according to WHO criteria for acceptability gave consistent data that demonstrated an attenuated phenotype for the YF 17DD 993FB013Z (13Z) vaccine batch. It is concluded that the additional chicken embryo passage did not lead to any genetic change and the new working seed virus retained its attenuation for monkeys comparable to the 102/84 reference virus.