The isolation of chicken minisatellites is reported. Two charomid libraries were constructed from fractions of size-selected MboI-digested genomic DNA. A total of 5985 colonies were screened with the multilocus probes 33.6 and 33.15; 137 positives were obtained (2.35%). A total of 55 clones were tested against four unrelated chickens; 30 revealed variable single locus patterns. More detailed characterization was made of 15 probes, which were tested against four families comprising a total of 53 offspring. Of parent/offspring comparisons 51% were informative for segregation. No mutations were detected. Three minisatellites were linked on the same autosome, implying that these loci may occur in clusters, and one was Z chromosome-linked. Heterozygosity and allelic variability were measured in 67 individuals from several different strains and breeds. Mean heterozygosity ranged from 50 to 84%. Minisatellite loci appear to be highly variable in the chicken and should provide highly informative markers in intraspecific crosses in genome mapping studies.