A method to quantify the contribution of subpopulations to genetic diversity in the whole population was assessed using pedigree information. The standardization of between- and within-subpopulation mean coancestries was developed to account for the different coat colour subpopulation sizes in the Spanish Purebred (SPB) horse population. The data included 166264 horses registered in the SPB Studbook. Animals born in the past 11 years (1996 to 2006) were selected as the ‘reference population’ and were grouped according to coat colour into eight subpopulations: grey (64 836 animals), bay (33 633), black (9414), chestnut (1243), buckskin (433), roan (107), isabella (57) and white (37). Contributions to the total genetic diversity were first assessed in the existing subpopulations and later compared with two scenarios with equal subpopulation size, one with the mean population size (13 710) and another with a low population size (100). Ancestor analysis revealed a very similar origin for the different groups, except for six ancestors that were only present in one of the groups likely to be responsible for the corresponding colour. The coancestry matrix showed a close genetic relationship between the bay and chestnut subpopulations. Before adjustment, Nei’s minimum distance showed a lack of differentiation among subpopulations (particularly among the black, chestnut and bay subpopulations) except for isabella and white individuals, whereas after adjustment, white, roan and grey individuals appeared less differentiated. Standardization showed that balancing coat colours would contribute preserving the genetic diversity of the breed. The global genetic diversity increased by 12.5% when the subpopulations were size standardized, showing that a progressive increase in minority coats would be profitable for the genetic diversity of this breed. The methodology developed could be useful for the study of the genetic structure of subpopulations with unbalanced sizes and to predict their genetic importance in terms of their contribution to genetic variability.