The effect of loading, transportation, lairage and slaughter conditions on bleed-out times, behavioural and physiological responses of Nguni and non-descript steers reared extensively on natural pastures was investigated. Twenty Nguni (NG) and 20 non-descript (ND) steers were loaded and transported 120 km from the farm to the abattoir in two groups (TG1 and TG2), each comprised of both genotypes. Some environmental conditions and steer behavioural responses were monitored during on- and off-loading, transportation, lairage, and stunning. Trained observers recorded the posture of the steers during transportation, time-budgets during lairage, avoidance-related behaviour, and vocalization scores at stunning were recorded. The steers were slaughtered in four groups (SG1, SG2, SG3 and SG4) and the number of attempts to stun each steer was recorded. Blood samples were collected from each steer during exsanguination for cortisol, glucose and lactate analysis. It took less time to load (370 s) and off-load (602 s) TG1 than TG2 (420 s and 782 s, respectively). All steers were standing throughout transportation and during the lairage observation period. Avoidance-related behaviour and vocalization in the stunning box were not influenced by genotype. The TG1 steers showed more avoidance behaviour (63.2%) and higher cortisol (140.6 ± 14.50 nmol/L) and lactate (12.4 ± 0.83 nmol/L) levels than TG2 (23.9%; 92.8 ± 15.38; 9.0 ± 0.88, respectively). All SG2 steers showed minimal avoidance behaviour with higher cortisol (175.9 ± 17.24 nmol/L) and lactate (13.5 ± 1.12 mmol/L) levels than other groups. Generally, cortisol and lactate levels were positively correlated (r = 0.70). The 5% vocalization recorded was observed from ND steers, TG1 and SG2. In conclusion, steers of different genotypes displayed similar behavioural and physiological responses to identical pre-slaughter conditions that they were exposed to.