© 2015 Elsevier Inc. Sheep of calm or nervous temperament differ in their physiological (cortisol secretion) and behavioural (motor activity) responses to stressors, perhaps due to variation in genes that regulate glucocorticoid synthesis or brain dopamine activity. Using ewes that had been selected over 20 generations for nervous (n = 58) or calm (n = 59) temperament, we confirmed the presence of a polymorphism in a gene specifically involved in cortisol production (CYP17), and identified polymorphisms in three genes specifically associated with personality and behavioural traits: dopamine receptors 2 and 4 (DRD2, DRD4), and monoamine oxidase A (MAOA). The calm and nervous lines differed in their frequencies of CYP17 SNP628 (single nucleotide A-G mutation at position 628) and DRD2 SNP939 (single nucleotide T-C mutation at position 939), but not for other SNPs detected in DRD2 or MAOA. In a second experiment, we then genotyped a large, non-selected flock of ewes for DRD2 SNP939 and CYP17 SNP628. Responses to the 'arena' and 'isolation box' challenges were associated with the DRD2 SNP939 genotype and the response to ACTH challenge was associated with the CYP17 SNP628 genotype. We conclude that, for sheep, a combination of the DRD2 SNP939 C allele and the CYP17 SNP628 A/A genotype could be used as a genetic marker for nervous temperament, and that a combination of DRD2 SNP939 T/T and CYP17 SNP628 G/G could be used as a genetic marker for calm temperament.