Twinning in dairy cattle has been associated with many negative health andreproductive events that cause economic loss to the producer. Reports havesuggested that twinning rates are increasing and that there may be a positiverelationship between milk production and twinning frequency. Putativequantitative trait loci (QTL) for twinning and ovulation rate on bovinechromosomes 5, 7, 19 and 23 have been previously identified in otherpopulations. The objective of this study was to detect and possibly confirm theexistence and effects of these QTL in the North American Holstein population.Half-sib families of 20 North American Holstein sires with above averagetwinning rate predicted transmitting abilities (PTA) comprised the samplepopulation under investigation. Twinning rate PTA values had been estimated fromcalving data. DNA extracted from semen samples was analysed using 45-61microsatellite markers across the four chromosomes. Marker heterozygosity of thepatriarchs averaged 62%. Evidence of twinning QTL was found in multiple familieson chromosomes 5, 7 and 23 and in one family on chromosome 19. Four of the siresformed one three-generation family: one sire and three half-sib sons with sonsof their own. This extended family was analysed with additional markersconfirming a twinning QTL of significant size on chromosome 5.