Prenatal exposure to the estrogenic endocrine disruptor Diethylstilbestrol (DES), a prescribed drug given to millions of pregnant women worldwide, led to multiple reproductive effects in the exposed offspring. We have previously shown that DES exposure causes transgenerational impacts on female mice, affecting fertility, timing of puberty and anogenital distance. To determine the mechanism behind these transgenerational impacts, we investigated DNA methylation in oocytes from three generations of DES exposed mice. Female mice were exposed to DES during gestation and oocytes collected from control, F1, F2 and F3 DES exposed generations. Bisulfite converted libraries were generated using the post bisulfite adaptor tagging (PBAT) method. Whole genome bisulfite sequencing analysis was conducted to identify differentially methylated regions (DMRs) between treatment and control oocytes. We identified 140 DMRs that were common across F1, F2 and F3 oocytes exposed to a high dose of DES, and 80 DMRs that were common in F1, F2 and F3 oocytes when exposed to a low dose. Genes associated with these DMRs are involved in female infertility, breast cancer, ovarian disorders and metabolic function, phenotypes all previously described both in our DES mouse model and in human DES descendants. These results reveal a direct correlation between prenatal DES exposure, transgenerational changes in the female germ line methylome and phenotypes observed in DES descendants. These results provide insights into the mechanisms through which estrogenic endocrine disruptors are impacting human fertility and reproductive health.