The genome segments of two electrophoretically distinct variants of bluetongue virus (BTV) Serotype 2 (Ona A and Ona B) from the U.S.A. were analyzed by double-dimension gel electrophoresis of RNase T1 produced oligonucleotides. Segments 1, 4, 5, 6, 7 and 10 were examined individually after separation by SDS-PAGE; and Segments 2 and 3, and 8 and 9, which were difficult to resolve, were fingerprinted as pairs. The Ona A and Ona B strains appeared to be closely related since corresponding segments were comparable, sharing 53-89% of the large oligonucleotides counted. Since the strains with the Ona A electropherotype preceded Ona B infection in Florida, U.S.A. and since Ona A was indistinguishable from the early African isolate of Serotype 2, Ona B was thought to be a variant of an Ona A strain. These data tend to support the hypothesis that Ona B could have evolved from Ona A as the result of point mutations or genetic drift.