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Analysis of variation at microsatellite DNA loci is widely used in studies of parentage1, linkage2 and evolutionary history^3–5. The utility of microsatel I ites is primarily due to high levels of allelic diversity, believed to reflect mutation rates orders of magnitude higher than base pair substitutions at single-copy genes. For humans^6–14, mice^15–16, rats¹⁷ and pigs¹⁸, microsatellite mutation rates have been estimated at 10⁻³–10⁻⁵. However, a recent study comparing microsatellite variation in humans with non-human primates suggests that microsatellite mutation rates may vary considerably across taxa^19–20. We measured mutation rates of 24 microsatellite loci in mutation accumulation lines of Drosophila melanogaster. Surprisingly, only a single mutation was detected after screening 157,680 allele-generations, yielding an estimated average mutation rate per locus of 6.3 × ¹⁰⁻⁶, a mutation rate considerably lower …