Technical Abstract: Grape phylloxera, Daktulosphaira vitifoliae Fitch is a devastating pest of grapevines (Vitaceae). The pest causes significant losses in grape production in California as well as throughout the world. A subtractive-based hybridization strategy was used to construct microsatellite enriched genomic libraries. Fifty loci were identified for primer design and 19 produced good PCR products, seven of which reliably detected polymorphisms across the 32 phylloxera populations tested. These seven SSR loci were used to distinguish genetic diversity in California and European phylloxera collections. The results confirm the utility of these loci for analyzing genetic diversity, finger-printing strains, and studying host associations. A significant deviation from the Hardy-Weinberg equilibrium for the tested California populations suggests that parthenogenesis is perhaps the primary, if not only, reproductive system in California. The data also support the hypothesis that the levels of genetic diversity and the host adaptation found in California populations are likely the result of multiple introductions and host selection. The data therefore suggests quarantine control is crucial in regulating import/exchange grape plant materials. Understanding genetic diversity and reproductive biology of the pest will help design and develop effective strategies for the pest management.