BACKGROUND: Mutations to STR alleles are not well understood in terms of the mechanism(s) underlying such mutations or their relative frequency among different alleles at the locus. STUDY DESIGN AND METHODS: A cytosine/thymosine (C/T) polymorphism was discovered 13 nucleotides upstream from the 5′-end of the tandem array of the D5S818 locus (-13SNP). The -13SNP coincidentally creates a restriction site polymorphism for the restriction endonuclease SnaBl that is tightly linked to the tandem array of D5S818. Forty D5S818 addition/deletion mutations in the tandem array were characterized with RFLP analysis with SnaBl. RESULTS: Among 40 mutations studied, 34 (∼85%) were of paternal origin, 1 (∼3%) was of maternal origin, and 5 (∼13%) had an unclear lineage of origin. In 26 cases where the magnitude of the repeat change could be determined, 23 (88%) involved changes of a single repeat unit, whereas 3 (12%) involved changes of 2 or more repeats. The number of additions to the tandem array was roughly equal to the number of deletions from the tandem array. In 19 instances it was possible to identify the parental allele that underwent the mutation. Alleles 13 and 14 were prone to mutation, whereas Allele 11 was resistant. Thus, there is an unequal sensitivity to mutation among D5S818 alleles. CONCLUSIONS: The use of the linked -13SNP marker has revealed several features of mutations at the D5S818 locus: 1) Single repeat changes are the most commonly observed. 2) Mutations are more likely in the paternal lineage. 3) Not all alleles undergo mutation with equal frequency.