Yellow fever (YF) remains a threat to human health in tropical regions of Africa and South America. Live-attenuated YF-17D vaccines have proven to be safe and effective in protecting travellers and populations in endemic regions against YF, despite very rare severe reactions following vaccination - YF vaccine-associated viscerotropic disease (YEL-AVD) and neurological disease (YEL-AND). We describe the generation and selection of a live-attenuated YF-17D vaccine candidate and present its preclinical profile. Initially, 24 YF-17D vaccine candidate sub-strains from the Stamaril® and YF-VAX® lineage were created through transfection of viral genomic RNA into Vero cells cultured in serum-free media to produce seed lots. The clone with the 'optimal' preclinical profile, i.e. the lowest neurovirulence, neurotropism and viscerotropism, and immunogenicity at least comparable with Stamaril and YF-VAX in relevant animal models, was selected as the vaccine candidate and taken forward for assessment at various production stages. The 'optimal' vaccine candidate was obtained from the YF-VAX lineage (hence named vYF-247) and had five nucleotide differences relative to its parent, with only two changes that resulted in amino acid changes at position 480 of the envelope protein (E) (valine to leucine), and position 65 of the non-structural protein 2A (NS2A) (methionine to valine). vYF-247 was less neurovirulent in mice than Stamaril and YF-VAX irrespective of production stage. Its attenuation profile in terms of neurotropism and viscerotropism was similar to YF-VAX in A129 mice, a 'worst case' animal model lacking type-I IFN receptors required to initiate viral clearance. Thus, vYF-247 would not be expected to have higher rates of YEL-AVD or YEL-AND than Stamaril and YF-VAX. In hamsters, vYF-247 was immunogenic and protected against high viremia and death induced by a lethal challenge with the hamster-adapted Jimenez P10 YF virus strain. Our data suggests that vYF-247 would provide robust protection against YF disease in humans, similar to currently marketed YF vaccines.