Author Summary Microsatellites are short tandem repeat DNA sequences located throughout the human genome that display a high degree of inter-individual variation. This characteristic makes microsatellites an attractive tool for population genetics and forensics research. Some microsatellites affect gene expression, and mutations within such microsatellites can cause disease. Interruption mutations disrupt the perfect repeated array and are frequently associated with altered disease risk, but they have not been thoroughly studied in human genomes. We identified interrupted mono-, di-, tri- and tetranucleotide MSs (iMS) within individual genomes from African, European, Asian and American population groups. We show that many iMSs, including some within disease-associated genes, are unique to a single population group. By measuring the conservation of microsatellites between human and chimpanzee genomes, we demonstrate that interruptions decrease the probability of microsatellite mutations throughout the genome. We demonstrate that iMSs arise in the human genome by single base changes within the DNA, and provide biochemical data suggesting that these stabilizing changes may be created by error-prone DNA polymerases. Our genome-wide study supports the model in which iMSs act to stabilize individual genomes, and suggests that population-specific differences in microsatellite architecture may be an avenue by which genetic ancestry impacts individual disease risk.