The inheritance of mitochondrial and chloroplast genomes does not follow Mendelian laws, but proceeds by vegetative segregation. Most organisms show organelle homoplasmy, which is probably produced and maintained during sexual reproduction. We have tested the effect of prolonged vegetative multiplication in the maintenance of mitochondrial homoplasmy and the generation of heteroplasmy in cultivated olive trees, Olea europaea L. Seven trees, each representing a different variety of olive, were analysed by the study of an intergenic spacer region of the mitochondrial genome. A very high level of heteroplasmy was detected in all cases. We found multiple genome variants of the sequence analysed. The frequency of genomes with no changes in the spacer region was 11.5%. This means that 88.5% of genomes contain at least one change. The same spacer mitochondrial region was sequenced in several clones from four olive trees of a second generation of sexually reproduced trees. In these trees, many clones were identical and had no changes, which represents a clear reduction of the heteroplasmy (p < 0.001). Therefore, this work supports the relevance of the role of sexual reproduction in the maintenance of mitochondrial homoplasmy and also shows that mutations accumulate in a non-coding sequence of the mitochondrial genome when vegetative propagation is maintained for a long period of time.