Abstract Background Grape phylloxera (Daktulosphaira vitifoliae) is one of the world’s most important viticultural pests. However, the reproductive mode, genetic structure and host adaptation of phylloxera in various viticultural environments remains unclear. We examined reproductive mode and genetic structure of phylloxera by analyzing microsatellite makers across the samples from four vineyard-sites in California. Result The phylloxera populations in California are believed to have predominantly parthenogenetic reproduction. Therefore, genetic diversity of phylloxera is expected to be limited. However, this study showed relatively high levels of diversity in Napa and Yolo county populations with a large number of unique genotypes, average number of alleles (2.1 to 2.9) and observed heterozygosities (0.330 to 0.388) per vineyard-sites. Reproduction diversity index (G: N—unique genotypes versus number of samples) ranged from 0.500 to 0.656 among vineyard-sites. Both significant and non-significant P sex (probability of sexual reproduction) were observed among different repeated genotypes within each vineyard. Moreover, high variation of F IS was observed among different loci in each vineyard-site. Genetic structure analysis (UPGMA) and various measures of population differentiations (F ST, PCA, and gene flow estimates) consistently separated AXR#1 (Vitis vinifera x V. rupestris—widely planted in California during the 1960s and 1970s) associated populations from the populations associated with other different rootstocks. Conclusion Genetic diversity, G: N ratio, P sex and F IS consistently suggested the occurrence of both parthenogenetic and sexual reproduction in California populations. This study clearly identified two major groups of phylloxera obtained from various rootstocks, with one group exclusively associated with only AXR#1 rootstock, defined as “biotype B”, and another group associated with vinifera-based rootstocks, known as “biotype A”.