We report here nucleotide sequence and characterization of gene 11 from two tissue culture-adapted ATCC1 rhesus (RRV) and human (Wa) strains of rotavirus. Gene 11 sequence encodes a nonstructural protein, NSP5 and also encodes NSP6, from an out of phase open reading frame. Sequence of RRVATCC gene 11 represents the first report from a rhesus rotavirus which has more than 90% homology at the nucleotide and deduced amino acid sequence level with that of its closely related simian SA11 strain. The WaATCC gene sequence differed from that of published Wa (WaPub) at three nucleotide positions, one at 264 (GWa-Pub to AATCC-Wa), another a nucleotide insertion (A) at position 388 and the third, a deletion (A) at 416. The latter two changes in WaATCC NSP5 resulted in drastic amino acid changes within a 10-residue region (123–132) from VHVYQFQLTN in WaPub to DSCVSISTNH in WaATCC NSP5 protein. In this region, WaATCC NSP5 is closer to published sequences from other strains, suggesting the authenticity of the present sequence. The nucleotide difference between WaPub and WaATCC NSP5 sequences, however, did not affect the NSP6 deduced amino acid sequence, which is overall highly conserved among all the strains compared. Sequence-based phylogenetic analysis of gene 11 identified a high degree of conservation within the Group A rotaviruses. In addition, it also separated RRVATCC and WaATCC, suggesting rotavirus segregation by genogroup. An anti-NSP5 monoclonal antibody of SA11 recognized RRV NSP5 protein but not WaATCC NSP5 from the infected cells, further supporting the phylogenetic segregation of RRVATCC and WaATCC strains based on their NSP5 coding sequence.