The nucleotide sequences of three regions of the genomes of 13 yellow fever (YF) virus isolates were determined to define genetic variation and evolution of the virus. Phylogenetic trees generated from sequences of either the 5' terminal 1320 nucleotides of the genome, 754 nucleotides from the NS4A and NS4B genes, or the 3' terminal 511 nucleotides were very similar and contained minor differences. Overall, these results suggested that there were at least four major genotypes of YF virus, including one in Central/East Africa, one in West Africa, and two in South America. Examination of the 3' noncoding region (3'NCR) showed that only West African strains had a 3'NCR of 511 nucleotides while strains from Central/East Africa and South America had shorter 3'NCRs (443-469 nucleotides) due to the absence of YF specific repeat sequences (RYFs). Central/East African strains have two RYFs and West African strains have three RYFs while South American strains only have one copy of the RYF. It is speculated that duplication of the RYF took place in West Africa subsequent to the presumed introduction of YF virus into South America. Thus, both tick-borne (Mandl et al., J. Virol. 65, 4070-4077, 1991 and Wallner et al., Virology 213, 169-178, 1995) and mosquito-borne flaviviruses have variable 3'NCRs.