The nucleotide sequence of the S RNA segment of the Oropouche (ORO) virus prototype strain TRVL 9760 was determined and found to be 754 nucleotides in length. In the virion-complementary orientation, the RNA contained two overlapping open reading frames of 693 and 273 nucleotides that were predicted to encode proteins of 231 and 91 amino acids, respectively. Subsequently, the nucleotide sequences of the nucleocapsid genes of 27 additional ORO virus strains, representing a 42 year interval and a wide geographical range in South America, were determined. Phylogenetic analyses revealed that all the ORO virus strains formed a monophyletic group that comprised three distinct lineages. Lineage I contained the prototype strain from Trinidad and most of the Brazilian strains, lineage II contained six Peruvian strains isolated between 1992 and 1998, and two strains from western Brazil isolated in 1991, while lineage III comprised four strains isolated in Panama during 1989.