Yellow fever (YF) is a serious public health problem in Bolivia since at least the 19th century. Surprisingly, very limited information has been made available to date regarding the genetic characterisation and epidemiology of Bolivian YF virus (YFV) strains. Here, we conducted the genetic characterization of 12 human isolates of YFV collected in Bolivia between 1999 and 2008, by sequencing and analysis of two regions of the viral genome: a fragment encoding structural proteins "PrM" (premembrane and envelope) and a distal region "EMF," spanning the end of the virus genome. Our study reveals a high genetic diversity of YFV strains circulating in Bolivia during the last decade: we identified not only "Peruvian-like" genotype II viruses (related to previously characterized Bolivian strains), but also, for the fist time, "Brazilian-like" genotype I viruses. During the complete period of the study, only cases of "jungle" YF were detected (i.e., circulation of YFV via a sylvatic cycle) with no cluster of urban cases. However, the very significant spread of the Aedes aegypti mosquito across Bolivian cities threatens the country with the reappearance of an urban YFV transmission cycle and thus is required a sustained epidemiological surveillance.