A high degree of inbreeding has been reported to negatively impact semen quality in Friesian horses and Shetland ponies. Both breeds are characterized by a closed studbook, small population size, and high incidence of inbreeding. The Dutch Warmblood studbook (KWPN: Koninklijk Warmblood Paardenstamboek Nederland) is a much larger studbook with two distinct populations: the KWPN-Riding horses, managed as an 'open' studbook, and the KWPN-Harness horses, representing a much smaller subpopulation within the KWPN breed and managed as an 'almost closed' studbook. It was recently reported that the degree of inbreeding in KWPN-Harness horses has increased in recent decades due to the small gene pool; however, the degree of inbreeding is still lower than that of Friesian horses and Shetland ponies. We hypothesized that a high or rising degree of inbreeding might negatively impact semen quality. In the present study, we retrospectively compared semen quality parameters of stallions from four different breeds or types (Friesian Horses, Shetland Ponies, KWPN-Riding horses, and KWPN-Harness horses), each reported with different degrees of inbreeding. Semen concentration, and percentages of motile, morphologically normal and live spermatozoa, and the total number of morphologically normal, progressive motile spermatozoa per ejaculate (TNM) were analyzed for 2832 semen evaluations performed over a 15-year period. KWPN-Harness horses had a significantly lower sperm concentration, % motile spermatozoa and % live spermatozoa than KWPN-Riding horses but the % motile and % morphologically normal spermatozoa and TNM in both KWPN-Harness and KWPN-Riding horses were significantly higher than in Friesian horses and Shetland ponies. These results suggest a lower semen quality in KWPN-Harness than KWPN-Riding horses, potentially as a result of a higher coefficient of inbreeding. The negative trend observed in the KWPN-Harness horses may be a warning sign, and breeders or stud books should monitor the degree of inbreeding carefully to ...