Two 3-generation full-sibling reference families have been produced and form a unique resource for genetic linkage mapping studies in the horse. The F2 generations, now comprising 61 individuals, consist of 28- to 32-day-old embryos removed nonsurgically from two pairs of identical twin mares. The same stallion sired all F2s such that the two full-sibling families are half-sibling with respect to each other. The families are crossbred to maximize levels of heterozygosity and include Arabian, Thoroughbred, Welsh Cob, and Icelandic Horse breeds. Milligram quantities of DNA have been isolated from each embryo and from blood samples of the parents and grandparents. The families have been genotyped with 353 equine microsatellites and 6 biallelic markers, and 42 linkage groups were formed. In addition, the physical location of 85 of the markers is known, and this has allowed 37 linkage groups to be anchored to the physical map. The inclusion of dams in the genotyping analysis has allowed the generation of a genetic map of the X chromosome. Markers have been assigned to all 31 autosomes and the X chromosome. The average interval between markers on the map is 10.5 cM, and the linkage groups collectively span 1780 cM. The results demonstrate the benefits for horse linkage mapping studies of genotyping on these unique full-sibling families, which comprise relatively few individuals, by the generation of a comprehensive low-density map of the horse genome. (C) 2000 Academic Press.