We compared the sequence of an envelope protein gene fragment from 21 temporally distinct West Nile (WN) virus strains, isolated in nine African countries and in France. Alignment of nucleotide sequences defined two groups of viruses which diverged by up to 29%. The first group of subtypes is composed of nine WN strains from France and Africa. The Austral-Asian Kunjin virus was classified as a WN subtype in this first group. The second group includes 12 WN strains from Africa and Madagascar. Four strains harboured a 12 nucleotide in-frame deletion. The loss of the corresponding four amino acids resulted in the loss of the potential glycosylation site present in several WN strains. The distribution of virus subtypes into two lineages did not correlate with host preference or geographical origin. The isolation of closely related subtypes in distant countries is consistent with WN viruses being disseminated by migrating birds.