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Förster resonance energy transfer between identical chromophores (homo‐FRET) has been difficult to analyze since neither emission intensity nor lifetime changes with the occurrence of homo‐FRET. Herein we used a DNA scaffold to analyze homo‐FRET between pyrene moieties. The DNA scaffold was modified with two pyrenes and a quencher, anthraquinone. Homo‐FRET was detected by monitoring quenching of pyrene emission and the decrease in the fluorescence lifetime of pyrene. Homo‐FRET efficiencies could be calculated by excluding effects of hetero‐FRET. The experimentally determined efficiencies showed an excellent agreement with Förster theory. These results will inform design of novel molecular probes and light‐harvesting antennae. [ABSTRACT FROM AUTHOR]