Eucalyptus urophylla is an important species of the genus Eucalyptus that is used across the world, especially in Brazil. It is used for pulp and paper production, and charcoal production for iron and steel industry, thus making the development of biotechnological approaches essential for the breeding programs of this species. The polyploidy induction is one such approach that may introduce phenotypic characteristics of interest to the market and industry. This is the first study that shows the production, evaluation, and development of tetraploids (4x = 44) from a clone of E. urophylla. For this, the shoot apical meristems were cultured for elongation using in vitro conditions for 15 with 5 or 10 µM colchicine, trifluralin, or oryzalin. Following this, every 30 days, the elongated plantlets were transferred to JADS medium for multiplication using tufts as explants. Thereafter, four tufts containing six to eight buds each were inoculated in flasks followed by six subcultures and were then transferred to the greenhouse for acclimatization. The experiment was performed in a completely randomized design in a factorial scheme (3x2+1: antimitotic substance x concentrations + control), with 12 replicates. Analysis of nuclear DNA content and chromosome counting by flow cytometry, photosynthetic pigment quantification, stomatal size and density, survival, and rooting were performed for 110 days after acclimatization. Data were submitted for analysis of variance followed by Scott & Knott’s test (P ≤ 0.05). As a result, we observed that the oryzalin and trifluralin treatments were efficient in the induction of tetraploid plants, while colchicine proved to be ineffective. Also, trifluralin treatment at 5 µM provided a higher polyploidy induction (18.2% tetraploids) in comparison to the others. Diploid plants displayed 1.41 pg of nuclear DNA content with 22 chromosomes, while tetraploid plants showed 2.86 pg with 44 chromosomes. In conclusion, the tetraploid plants, as compared to the diploids (control), were found to show higher chlorophyll content, larger but fewer stomata, and lower rooting and survival, which can be mitigated nonetheless through silvicultural techniques. This study shall bring new perspectives for the breeding programs of E. urophylla