Eighteen strains of Rift Valley fever (RVF) virus collected over a period of 38 years and isolated from diverse localities in Africa and from various hosts (human, animal and arthropod) were investigated by RT-PCR followed by sequencing of the NS(S) protein coding region. This region was chosen to analyse variability because, in contrast to the N protein, the NS(S) protein differs in various phleboviruses and there exists an RVF virus (clone 13) in which 70% of the NS(S) ORF is deleted, suggesting that this sequence is under a weak selective pressure. Sequence data indicated that percentage divergence among isolates ranged from 0 to 9.6% at the nucleotide level and from 0 to 9.5% at the amino acid level. Phylogenetic analysis based on the NS(S) gene revealed two major lineages: Egyptian and sub-Saharan. This led to the establishment of the relatedness between strains and insights into the NS(S) protein, the function of which is still undetermined. Alignment of the deduced amino acid sequences indicated that the cysteine residues are conserved, as are several motifs representing potential phosphorylation sites.