The complete nucleotide sequence of rice stripe virus (RSV) segment 3 shows that it has two open reading frames, one in the viral-complementary sequence, which codes for the nucleocapsid protein, and the other in the viral-sense sequence. The non-coding region between the ambisense genes in RSV segment 3 contains several U and A tracts, as do the ambisense S segments of phleboviruses and uukuviruses. As we have previously shown that RSV segment 4 has an ambisense nature, this is the first instance of a virus containing two ambisense segments.