We demonstrate the influence of spectral linewidths of individual donor-acceptor couples on energy transfer efficiency in semiconductor nanocrystal-DNA-organic dye conjugates. Temperature-dependent single molecule and ensemble spectroscopy data are analyzed using the Forster theory within the macroscopic and microscopic approaches. The results obtained evidence on the importance of the spectral overlap between emission of a single donor and absorption of a single acceptor in its close vicinity, which determines the microscopic resonance and transfer efficiency between individual neighbors. This realization poses important implications on the applicability of ensemble spectral overlap for the analysis of distance dependencies of nanoscopic objects. (C) 2009 American Institute of Physics. [doi:10.1063/1.3242015]