Thyroid scanning using technetium-99m (99mTc) is the gold standard for diagnosing feline hyperthyroidism. In cats with an overactive thyroid, a thyroid scan is the most appropriate imaging technique to detect and localize any hyperfunctional adenomatous thyroid tissue. In this study, the pharmacological properties of the Technekitty injection (Tc-99m), developed as a diagnostic agent for feline hyperthyroidism using 99mTc as an active ingredient, were tested in FRTL-5 thyroid follicular cell line and ICR mice. The percentage of cell uptake of the Tc-99m in FRTL-5 thyroid cells was 0.182 ± 0.018%, which was about 6 times higher compared to Clone 9 hepatocytes. This uptake decreased by 38.2% due to competitive inhibition by iodine (sodium iodide). In tissue distribution tests by using ICR mice, the highest distribution was observed in the liver, kidneys, spleen, lungs, and femur at 0.083 hours after administration, and this distribution decreased as the compound was excreted through the kidneys, the primary excretory organ. Maximum distribution was confirmed at 1 hour in the small intestine, 6 hours in the large intestine, and 2 hours in the thyroid gland. Additionally, the total amount excreted through urine and feces over 48 hours (2 days) was 78.80% of the injected dose, with 37.70% (47.84% of the total excretion) excreted through urine and 41.10% (52.16% of the total excretion) through feces. In conclusion, the Tc-99m has the same mechanism of action, potency, absorption, distribution, metabolism, and excretion characteristics as 99mTc used for feline hyperthyroidism in the United States, Europe, and other countries, because the Technekitty injection (Tc-99m) contains 99mTc as its sole active ingredient. Based on these results, the Technekitty injection (Tc-99m) is expected to be safely used in the clinical diagnosis of feline hyperthyroidism.