The genetic structure and level of diversity were assessed across 6 riding-horse breeds raised in Spain and born between 1989 and 2012 (Anglo-Arab, AA; Arab Purebred, PRA; Spanish Purebred, PRE; Menorca Purebred, PRMe; Spanish Sport horse, CDE; Spanish Trotter horse, TRO). The impact of selection on molecular diversity over time looking at 2 different cohorts in Crossbreds (AA, CDE and TRO) vs Purebreds (PRA, PRE and PRMe) has been also analysed. A total of 17 microsatellite loci were analysed in 2,530 horses from the cohort 1 (C1, born between 1989 and 2000) and 10,102 horses from the cohort 2 (C2, born between 2001 and 2012). The mean number of alleles per locus was the highest in C2 of TRO horses (11.76) and the lowest in C1 of PRMe breed (7.71). The lowest global values for the expected heterozygosity were found within Purebreds (0.71–0.76) compared to Crossbreds (0.76–0.78). For C2, the effective population size values based on linkage disequilibrium (from 4.9 in PRA to 668.3 in PRE) were higher than those computed for C1 (from 3.3 in PRA to 204.1 in CDE). Genetic distances and structure analysis showed that a significant amount of genetic variation is maintained due to an increase in the genetic uniformity across generations in Pure and Crossbreds. PRA, CDE and AA breeds seem to be the most genetically related, and for PRE, PRMe and TRO, both cohorts appeared closely related. In all populations, the genetic variability and effective population size have increased over time. Thus breeding policies have been properly managed to preserve diversity levels. Regarding the intense impact of selection in Purebreds across generations shown through the differences in the structure of the populations, no loss of genetic variability is expected in a short-term, although an extremely high degree of similarity and homogeneity between individuals of Purebreds compared to Crossbreds was shown.