Single molecule fluorescence resonance energy transfer has been extensively used to measure distance changes and kinetics in various biomolecular systems. However, due to complications involving multiple de-excitation pathways of the dyes, the absolute inter-dye distance information has seldom been recovered. To circumvent this we directly probe the relative variations in the quantum yield of individual fluorophores. B-DNA was used as a scaffold to position the donor (Cy3 or TMR) at precise distances from the acceptor (Cy5) within the Forster radius. We found that the variation in the Cy3 quantum yield is approximately 5 times larger than that of TMR. By taking into account the molecule-to-molecule variability in the acceptor/donor quantum yield ratio, the apparent fluorescence resonance energy transfer efficiencies were scaled to yield the theoretical values. We obtained very good agreement with a physical model that predicts distances along B-DNA.