Abstract Background The germline genetic events underpinning medulloblastoma (MB) initiation, and therefore the ability to determine who is at risk, are still unknown for the majority of cases. Microsatellites are short repeated sequences that make up ~3% of the genome. Repeat lengths vary among individuals and are often nonrandomly associated with disease, including several cancers such as breast, glioma, lung, and ovarian. Due to their effects on gene function, they have been called the “tuning knobs of the genome.” Methods We have developed a novel approach for identifying a microsatellite-based signature to differentiate MB patients from controls using germline DNA. Results Analyzing germline whole exome sequencing data from a training set of 120 MB subjects and 425 controls, we identified 139 individual microsatellite loci whose genotypes differ significantly between the groups. Using a genetic algorithm, we identified a subset of 43 microsatellites that distinguish MB subjects from controls with a sensitivity and specificity of 92% and 88%, respectively. This microsatellite signature was validated in an independent dataset consisting of 102 subjects and 428 controls, with comparable sensitivity and specificity of 95% and 90%, respectively. Analysis of the allele genotypes of those 139 informative loci demonstrates that their association with MB is a consequence of individual microsatellites' genotypes rather than their hypermutability. Finally, an analysis of the genes harboring these microsatellite loci reveals cellular functions important for tumorigenesis. Conclusion This study demonstrates that MB-specific germline microsatellite variations mark those at risk for MB development and suggests mechanisms of predisposition.