In Olea europaea L. subsp. europaea, tetraploid genotypes do not exist in nature. Herein, we report the first example of selection of tetraploid olive plants, obtained by combining physical mutagenesis and biotechnology techniques. Stable tetraploid (4n) and diploid (2n) genotypes were isolated in vitro through shoot-tip fragmentation of two mixoploid mutants derived from the gamma irradiation of self-incompatible Leccino and self-compatible Frantoio cultivars. In this study, the stable mutants FRM5-4n, FRM5-2n, LM3-4n, and LM3-2n were characterized in the field for vegetative and reproductive behavior with the aim to use them as varieties or dwarfing rootstocks. The stable 4n genotype of Leccino acquired self-fertility whereas the 4n Frantoio maintained it. A high and constant yield was showed by LM3-2n during 9 years of observation, maintaining the same oil quality as the Leccino wild type (wt). Moreover, the LM3-2n acquired the capacity to be intercompatible with the diploid mutant Leccino dwarf (LD) and with the Leccino wt. This acquired property would allow for a reduction of heterozygosity in the offspring, if crossed with each other for some generations and with the Leccino wt, because it is a sort of self-fertilization. When used as rootstocks, both 4n and 2n Leccino mutants proved to be very effective for reducing the scion size of the high-vigor Canino cultivar, which is well known for its excellent extravirgin oil. Finally, it was demonstrated that the self-grafting of vigorous cultivar caused a reduction in plant size, thus suggesting that it is possible to produce semidwarf plants from vigorous genotypes to consider them in high-density olive orchards.