Gene mutations influencing melanocytes also impact on physiological and behavioural functions. In this study, we investigated their association with four different coat colours in the Pura Raza Español (PRE) horse using morphological traits and molecular datasets. Four different subpopulations were identified according to individual coat colour: grey, bay, chestnut and black. Coat colour significantly associated with morphological measurements. Observed and expected heterozygosity values were low in grey compared with the other three subpopulations, suggesting the presence of unique ancestral alleles probably arisen by genetic drift and selection mechanism effects. Nei's distance demonstrated a clear division among subpopulations, the grey being the most divergent group. Gene flow estimates were similar, showing the lowest values in grey. Divergence times among subpopulations assessed with the average square distance suggested that grey was the original PRE population which diverged from bay, chestnut and black. Our results also demonstrated a clear morphological differentiation according to coat colour. The close genetic structure of bay and chestnut PRE subpopulations and the clear differences in most morphological traits of grey and chestnut PRE mares would suggest the pleiotropic effect of genomic regions determining coat colour in horses. However, further analysis including genomic information would be necessary to elucidate the mechanisms involved.