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Background: According to the literature review, this is the first study investigating tear production (TP) and intraocular pressure (IOP) in the Pygoscelis penguins living in their natural habitat. The study aimed to establish normal values for standard ocular tests in the genus Pygoscelis, namely, the Adélie (Pygoscelis adeliae), gentoo (Pygoscelis papua), and chinstrap (Pygoscelis antarctica) penguins, in four different islands of Antarctica. Sampling was made by specifically using the left eye of the penguins. The Schirmer's tear test type I (STT-I) and the Tonovet® (rebound tonometer) were used to measure the TP and the IOP, respectively. Results: The mean TP and IOP values of 129 Adélie, chinstrap, gentoo, and 120 adult Adélie, gentoo penguins were determined as 10.2 ± 4.0 mm/min and 38.9 ± 13.2 mmHg, respectively. No statistical difference was detected between the penguin species for the mean IOP values, while the difference was determined in all the locations. However, statistical differences in the mean TP values were determined between all locations. Conclusion: The results of this study provide a reference range of Schirmer's tear test (STT) and IOP values in Pygoscelis penguins and show that the IOP is significantly affected by locations. This result can be attributed to the harsh climatic conditions of the Antarctic Peninsula that change very quickly. The described data may help diagnose clinical pathological findings in Pygoscelis penguins. The STT and rebound tonometry appears to be safe and reproducible methods in Pygoscelis penguins, as the results were obtained quickly and were well tolerated by the birds. Based on our results, we propose that similar studies can be initiated in crowded colonies of three penguin species of this genus on the Antarctic Peninsula, the southern Shetland Islands, and other frequently visited islands in Antarctica. Highlights: • This study is the first to investigate tear production (TP) and intraocular pressure (IOP) in Pygoscelis penguins. • TP value was obtained by Schirmer's tear test type I (STT- I), while the IOP was measured with the help of Tonovet® (rebound tonometer). • No clinical macroscopic findings affecting the eyelids, third eyelid, cornea, or ocular eye surface were found during the clinical examination of the penguins. • There was no difference in TP values between species of this genus and locations. There was no statistically significant difference between species in IOP mean values. However, a significant difference was noticed among the locations. • This study indicated that IOP in Pygoscelis penguins was significantly affected by location. [ABSTRACT FROM AUTHOR]