Abstract The induction of polyploidy in Eucalyptus can lead to higher-yielding and more adapted clones, with better wood quality. After artificial polyploidization, it is necessary to certify the effectiveness of the process through cytogenetic analyses and anatomical trait evaluations. Thus, the aim of this study was to certify the chromosome duplication in artificially polyploidized clones of Eucalyptus grandis × Eucalyptus urophylla using cytogenetic analysis, flow cytometry, and leaf anatomy measurements. Chromosomal counts and DNA content estimation allowed the identification of artificially polyploidized clones and mixoploids. Polyploid clones had larger stomata diameter than diploids. The increase in stomata size was accompanied by lower stomatal density in most polyploid clones. The typical gigas effect of polyploidized plants was demonstrated by the expressive increase in leaf area in polyploid individuals compared to diploids. Our findings indicate that the use of polyploidy in Eucalyptus breeding is promising, allowing the discrimination of diploid, tetraploid, and mixoploid plants.