Glycine-rich RNA-binding proteins (GR-RBPs) play important roles in the post-transcriptional regulation. In this paper, 11 GR-RBPs genes (RGP-3) were isolated and sequenced from different tobacco varieties including cultivated tobacco and wild tobacco. Sequence homology, phylogenetic analysis and gene expression were discussed as emphases. Sequences alignment revealed that RGP-3 in different tobacco varieties show high homology, with 100% similarity between K326 and N. sylvestris and the lowest similarity of 88.1% between N. goodspeedi and N. repanda. There were 426 variation sites in RGP-3 genomic sequence, but 383 sites were located in introns. Phylogenetic trees revealed that K326 Hongda were evolutionarily closest to N. sylvestris, suggesting closest genetic relationship between N. tabacum and N. sylvestris. Expression analysis revealed that RGP-3 in N. tabacum was strongly induced by water, weakly induced by SA, and unaffected by ABA treatment. These results will provide the basis for a wide cross to improve tobacco biotic and abiotic resistance.