A novel variant of Rice Tapetum Specific (RTS) promoter, RTS64 isolated from indica rice cv. IR64, differed from reported RTS promoter from IR54 (RTS54) in the nucleotide sequence as well as in number and distribution of cis-acting regulatory elements. The new promoter contains motifs implicated in strong anther-specific expression. RTS64 promoter showed enhanced expression in anther compared to reported RTS54 promoter as indicated by Gus reporter assay. This novel anther-specific promoter, thus, would be a better candidate in genetic engineering strategies that require a high level of anther-specific expression.