Some U.S. bioethanol plants use a blend of maize and sorghum grains as feedstocks depending on availability and price. Knowledge of the grain composition of milled feedstock is beneficial for maximizing process efficiency and for potential future regulatory requirements. To analyze feedstock composition, we developed a duplex real-time PCR assay based on TaqMan probes for the simultaneous analysis of flour blends. For sorghum, the method amplified a sequence within the F-Box domain-containing protein and for maize, the method amplified a sequence of the high mobility group a (HMGa) proteins. To acquire fluorescent signals simultaneously, FAM and TAMRA fluorescent probes were used to label the sorghum- and maize-specific probes, respectively. Results showed that the primers and probes were highly specific for either maize or sorghum. The real-time duplex PCR assay was validated on simulated flour binary mixtures and was high in the precision and accuracy for the sorghum PCR system. Results with the established method showed specificity and sensitivity in quantifying sorghum composition in bioethanol feedstock mixtures.