Intraocular pressure (IOP), an important risk factor for glaucoma, is a continuous trait determined by a complex set of genetic and environmental factors that are largely unknown. Genetic studies in laboratory animals may facilitate the identification of genes that affect IOP. We examined the use of the rebound tonometer for measuring IOP in non-anaesthetised birds, along with the device's robustness to alignment errors. Calibration curves were obtained by measuring the IOP of cannulated chicken eyes with the rebound tonometer over a range of pressures. To simulate different types of alignment errors that might be expected with measurement of IOP in alert chickens, for some calibrations the tonometer was positioned (1) at various distances from the cornea, (2) laterally displaced from the visual axis, or (3) angled away from the visual axis. In vivo measurements were taken on three-week-old alert chickens from a layer line, a broiler line, and a layer-broiler "advanced intercross line" (AIL) designed to facilitate QTL mapping. The rebound tonometer showed excellent linearity (R2=0.95-0.99) during calibration, as well as robustness to variation in the probe-to-cornea distance over the range 3-5mm and to lateral displacement over the range 0-2mm. However, the tonometer appeared less robust to off-axis misalignment over the range 0-20 degrees (P<0.05). Also, the slope of calibration curves sometimes differed between eyes (P<0.001), presumably reflecting differences in ocular structure. The IOP measured in non-anaesthetised three-week-old AIL chickens was 17.51+/-0.13 mmHg (mean+/-S.E.; N=105 birds). IOP was significantly associated with corneal thickness (P<0.05) and body weight (P<0.001) in a regression model. Replicate measurements were necessary in order to gauge IOP accurately in individual birds; a series of seven tonometry sessions over a 12-h period during the light phase of the light/dark cycle permitted IOP to be measured with a 95% CI of +/-0.7 mmHg. IOP did not differ significantly between the broiler and layer chicken lines which served as the progenitor lines for the AIL. In conclusion, the rebound tonometer permits rapid estimation of IOP in chickens and is well tolerated. The small alignment errors that are expected when taking measurements in non-anaesthetised animals are unlikely to affect accuracy. Since high IOP is a major risk factor for glaucoma, identifying QTL controlling IOP may offer future health benefits. However, our preliminary findings highlight several obstacles to mapping such QTL using the chicken advanced intercross line evaluated here.