Sterile triploid European graylings (Thymallus thymallus) could serve as an alternative to allochthonous stocking, potentially protecting native populations from genetic introgression. In this study, two-year-old triploid and diploid graylings were examined to assess their hematological and biochemical characteristics and to evaluate the development of their gonads. When compared to diploids, triploids exhibited elevated white blood cell counts, mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) by 5.66%, 162.68% and 207.57%, respectively. Moreover, the diameters of red blood cells and their nuclei were significantly higher in triploid graylings. In contrast, the red blood cell count, hemoglobin concentration (Hb), hematocrit and mean corpuscular volume (MCV) were lower in triploids by 64.82%, 5.80%, 70.16% and 14.49%, respectively. Most blood plasma biochemical indices showed no significant differences between specimens of different ploidies; however, triploids had a 21.96% higher level of triglycerides, while diploids had 3.74% more albumin. Additionally, the chloride concentration was 4.74% lower in triploids. Examined diploid males exhibited well-developed, sometimes asymmetrical testes and were actively spermiating. Triploid males were non-spermiating, and their testes were solid with varying morphology. Ovaries in diploid females contained mature oocytes, and in about 30% of the females, the body cavities were filled with ovulated eggs. In turn, the ovaries in triploid females were significantly reduced, usually transparent and lacked any oocytes. However, a longer study over a period exceeding two years needs to be performed to state unequivocally that triploid grayling females are sterile.