BackgroundMeasurement of the intraocular pressure (IOP) is a useful diagnostic tool in equine ophthalmology. Handheld tonometers, such as Tonovet and Tonovet Plus (rebound), Tono‐Pen AVIA Vet (applanation), and Kowa HA‐2 (applanation using the Goldmann methodology) are used to obtain IOP measurements in veterinary medicine.ObjectivesTo compare and evaluate the accuracy of four handheld tonometers in measuring IOP using different methodologies in healthy horses.Study designIn vivo experiment and cross‐sectional survey of healthy horses.MethodsIntraocular pressure was measured in 72 eyes of 36 horses. An in vivo study was conducted on sedated horses to compare the real IOP values obtained using manometry versus those obtained using tonometry, and a field study was conducted on unsedated healthy horses with normal eyes to measure the IOP values using different tonometers.ResultsIn the in vivo study, the mean IOP values using ocular manometry was 24.9 ± 4.0 mmHg (range, 20.0–30.0 mmHg). The mean IOP values using tonometry were: Tonovet, 25.7 ± 5.8 mmHg (range 19.5–33.0 mmHg); Tonovet Plus, 24.8 ± 7.1 mmHg (range 13.2–33.2 mmHg); Tono Pen AVIA Vet, 19.2 ± 4.7 mmHg (range 13.1–26.5 mmHg); and Kowa Ha‐2, 24.1 ± 1.2 mmHg (range 22.8–25.8 mmHg). In the field study, the IOP values were: Tonovet, 30.7 ± 5.6 mmHg (range 21.7–38.0 mmHg); Tonovet Plus, 29.6 ± 6.7 mmHg (range 16.2–38.6 mmHg); Tono‐Pen AVIA Vet, 27.3 ± 5.8 mmHg (range 14.6–37.1 mmHg); and Kowa HA‐2, 23.4 ± 2.2 mmHg (range 20.2–28.7 mmHg).Main limitationsThis study included only healthy horses and a limited number of animals in the in vivo study.ConclusionsThere was a strong correlation between the IOP values and manometry for all tonometers. IOP should be estimated using the same tonometer over time, and the bias of the tonometer used, such as overestimation (rebound tonometer) and underestimation (applanation tonometer), should be acknowledged. A normal reference value for each tonometer should be established in horses.