Vacancy-type defects in strained-Si layers deposited on Si0.75Ge0.25/graded-SiGe/Si structures were probed by using monoenergetic positron beams. The Doppler broadening spectra of the annihilation radiation and the lifetime spectra of the positrons were measured for samples before and after annealing (800–1050 °C). For an as-received sample, the defects in the strained-Si layer were identified as vacancy-type defects coupled with Ge. The mean open size of these defects was estimated to be close to that of a divacancy. The line-shape parameter, S, corresponding to the positron annihilation in the strained-Si layers decreased with increasing annealing temperature, but no large change in the positron lifetime was observed. From a comparison between the Doppler broadening profiles for the strained-Si films and those calculated using the projector augmented-wave method, it was found that the number of Ge atoms forming a complex by coupling with a defect increased with increasing annealing temperature. The number was estimated to be three or four after annealing at 1050 °C. Since the defect complexes were stable even after annealing at such a high temperature, the defects detected by the positrons could be part of chainlike vacancy clusters.