Background: NS1 gene of dengue virus produces a hexameric soluble NS1 protein that is the first viral protein to circulate in host circulation. The viral protein may interact with other human proteins; thus it can be hypothesized to be under selection pressure. Since the probability of molecular heterogeneity is likely in such cases, the present study was carried-out to evaluate the usefulness of the NS1 gene for genotyping of DENV-3 isolated during 2006 major dengue fever outbreak. Methods: Molecular subtyping was performed on eight DENV-3 isolates (sampled in 2006) by comparing with other NS1 sequences reported from India and other parts of the world. For comparison, a 390 bp region from amplified NS1 gene was selected. A phylogenetic tree was constructed by MEGA 3.1 software using Neighbor-Joining method with bootstrap value of 1000 replicates. DENV-3 prototype strain, H-87 was used to root the tree. Results: All the NS1 DENV-3 sequences segregated in 3 distinct genotypes as established on the basis of CprM and E genes in earlier studies; although genotype IV could not be shown due to non-availability of the sequences. Genotype I consisted of viruses isolated in Tahiti, Indonesia and Philippines isolated between 1978—98. Genotype II consisted of viruses from Thailand and Taiwan isolated between 1987—98. Genotype III was further divided in three clades: Indian clade consisting of NS1 2006 and 2005 Indian isolates; American clade consisting of isolates from Martinique and Brazil isolated between 1999—04; and the clade consisting of Singapore (2004—05) and Sri Lanka (2000) isolates. Conclusion: NS1 gene has been found to be useful in molecular subtyping of DEN-3 viruses, although the method has to be evaluated for other circulating dengue virus serotypes.