Background <p>Polyploidization plays a major role in citrus breeding, particularly in generating triploid hybrids through controlled crosses between diploid female and tetraploid male parents. However, limited information is available on how polyploidy affects pollen performance in citrus, and in particular, how temperature influences these processes. In this study, we compared pollen viability under field conditions and pollen tube growth at different temperatures in diploid and tetraploid genotypes, and assessed whether the observed differences were related to genomic instability caused by the tetraploidization treatment.</p> Results <p>Tetraploid `Clemenules´ and `Fortune´, both colchicine-derived, showed a higher proportion of collapsed pollen grains and reduced viability compared with their diploid counterparts, whereas the spontaneous tetraploid `Pineapple´ displayed values similar to those of the diploid. Pollen tube growth assays revealed slightly faster tube elongation in tetraploids, and notably, self-incompatible diploid pistils of `Fortune´ were successfully fertilized by pollen from its tetraploid form. Genotyping-by-sequencing (GBS) analyses of diploid and tetraploid plants revealed no chromosomal rearrangements or dosage alterations, suggesting that colchicine treatment did not induce large-scale genomic instability.</p> Conclusions <p>Our findings demonstrate that tetraploidy influences several aspects of citrus pollen performance, while genomic stability remains largely preserved in colchicine-induced genotypes. These results provide new insights into the reproductive biology of citrus polyploids under different temperature scenarios and reinforce the potential use of tetraploid parents in triploid breeding programs.</p>