According to literature review, this is the first study investigating tear production and intraocular pressure in pygoscelid penguins living in their natural habitat. This study also provides reference values for penguins kept under professional care at zoos and rehabilitation centres. This study aimed to establish normal values for standard ocular tests, including the Schirmer tear test (STT-1) and intraocular pressure test (IOP), for penguins belonging to the genus Pygoscelis (P), namely, the Adélie (Pygoscelis adeliae), gentoo (Pygoscelis papua) and chinstrap (Pygoscelis antarctica) penguins. Ophthalmic measurements were made from the Antarctic. In the left eye of each penguin, the amount of tear production (TP) was determined with the STT-1 and the IOP was measured using a Tonovet® rebound tonometer. No macroscopic findings affecting the eyelids, third eyelid, cornea or anterior eye camera were detected in the eyes of the examined penguins. The mean STT-1 and mean IOP values of 129 and 120 adult penguins were determined as 10.163 ± 4.054 mm/min and 38.852 ± 13.188 mmHg, respectively. A statistical difference at the level of p < 0.001 was determined between the islands for the mean IOP values. While no statistically significant difference was detected between the penguin species for the mean IOP values, between the locations was found to be significant (p < 0.001). Statistical differences at a level of p < 0.05 were determined for the mean TP values between the all locations and for the mean IOP values between the all locations ( p < 0.001) and only Lions Rump - Ardley I (p = 0.023). A statistical difference of p < 0.05 was detected between the chinstrap and gentoo for the mean STT-1 values. This study, which is aimed at reporting the first literature data in this field, has shown the need for further more detailed studies to elucidate the impact of different locations, daily time intervals and seasons on the STT and IOP values of penguins.