Observed and expected heterozygosity (H) levels for ten electrophoretic blood marker loci and expected H for seven red blood cell (RBC) anitgen/antibody loci were examined for 20 years in American Standardbred and 30 years in American Saddlebred horses. Standardbreds were classed by gait, Trotter and Pacer, and evaluated separately in most analyses. 4,404 Trotters and 12,271 Pacers were found to have statistically highly significant losses of mean total observed H through time for the ten electrophoretic loci (P<0.005), although in Trotters the loss was more extreme (P<<0.001). Loss of H in 5984 Saddlebreds was not significant (P=0.259). Correlations of RBC expected H through time showed decreases in all three groups.