► Group A rotaviruses are the leading cause of acute gastroenteritis in children. ► We analysed Australian rotavirus NSP2 gene sequences. ► Phylogenetic analysis shows conservation of Australian NSP2 gene sequences. ► NSP2 genes exhibited recombination, reassortment and interspecies transmission. The rotavirus non-structural protein NSP2 is one of the earliest and most abundant viral proteins produced during infection. This protein has multiple essential roles in the replication cycle involving RNA binding, viroplasm formation, helicase and can hydrolyse the γ-phosphate of RNA and NTPs acting as an RTPase and NTPase. In studying sequences from rotavirus strains isolated in Australia between 1984 and 2009, the NSP2 gene was seen to be highly conserved and clustered with defined NSP2 genotypes N1 and N2 according to the full genome based rotavirus classification system. Phylogenetic analysis indicated that NSP2 gene sequences isolated from Australian rotavirus strains formed four distinct lineages. Temporal variation was observed in several clusters during the 26 year period, with lineage D identified throughout the entire study period and lineage A only detected since 1999. Phylogenetic analysis and dendrograms identified NSP2 genes that exhibited reassortment between different virus VP7 genotypes, as well as a sequence from a human strain that grouped closely with the NSP2 genes of bovine rotavirus strains. This study also identified a sequence that fell between lineages and exhibited evidence of recombination, the first time that intergenic recombination has been detected in a NSP2 gene sequence. This study increases the understanding of the evolution mechanisms of NSP2 in view of improved vaccine design.