A hamster viscerotropic strain of yellow fever (YF) virus has been derived after serial passage of strain Asibi through hamsters. The parental Asibi/hamster p0 virus causes a mild and transient viremia in hamsters with no outward, clinical signs of illness. In contrast, the viscerotropic Asibi/hamster p7 virus causes a robust viremia, severe illness, and death in subadult hamsters. The genome of the hamster viscerotropic Asibi/hamster p7 virus has been sequenced and compared with the parental nonviscerotropic Asibi/hamster p0 virus identifying 14 nucleotide changes encoding only seven amino acid substitutions. The majority of these substitutions (five of seven) fall within the envelope (E) protein at positions Q27H, D28G, D155A, K323R, and K331R. These results support an important role for the E protein in determining YF virus viscerotropism.