We describe the development and application of an expressed sequence tag (EST)-derived restriction fragment length polymorphism (RFLP) marker for sugarcane elite genotypes which can be used for quantitative trait loci (QTL) tagging for sugar content. EST-derived RFLP markers for proteins involved in sucrose metabolism have been used in Southern analysis for mapping and gene tagging in elite sugarcane clones. A single dose marker, obtained from a sucrose synthase EST associated with sugar content at the alpha = 0.01 probability level, is presented for sugarcane breeding. Utilization of EST homologues to known genes for generation of molecular markers accelerated the identification of a QTL controlling an important trait-sugar content. Sugarcane bacterial artificial chromosome (BAC) clones hybridizing to the sucrose synthase EST were identified.