A relationship between dopamine and temperament has previously been described in human cases of dopaminergic dysfunction. Adjustment in temperament prior to disease manifestation can enable the early identification of individuals at risk of such conditions, and scope exists to extend this application of temperament alterations to cases of dopaminergic dysfunction in horses. A multivariate and mixed-methods approach utilising a questionnaire along with two inferred measurements of dopamine activity (Spontaneous Blink Rate [SBR] and Behavioral Initiation Rate [BIR]) were recorded from direct observation of animals (n = 99) to identify the potential relationship between dopamine and temperament in horses. Principal components analysis (PCA) of 36 temperament variables revealed nine Principal Components, including ‘Anxiety’ and ‘Docility’, which accounted for 72.4% of the total variance. Component scores were calculated and correlated with SBR and BIR utilising Spearman Rank Correlation Coefficient analysis. The component ‘Anxiety’ was found to have a significant positive relationship with SBR, whereas ‘Docility’ was observed to have a significant negative relationship with SBR. These results indicate a relationship between dopamine and temperament within the horse that is certainly worthy of further study. Potential mechanisms involving neural dopaminergic and GABAergic systems are presented, in addition to how such alterations could be utilised to probe for equine dopamine dysfunction pending future research.