In turtles with temperature-dependent sex determination (TSD), estradiol applied to eggs incubated at male-producing temperatures produces female hatchlings. In other vertebrate taxa, estradiol administration during embryogenesis often induces only temporary or incomplete feminization, and sensitivity to estradiol can vary among taxonomic families. In the present study, we treated eggs of Kinosternon scorpiodes and Sternotherus odoratus incubated at male-producing temperatures with estradiol-17β and examined reproductive morphology of offspring. Furthermore, to determine whether sex reversal resulting from estradiol application in turtles with TSD is lasting, we maintained juvenile K. scorpiodes in the lab for up to three years prior to dissection. We found normal ovarian morphology and Müllerian duct development in a significantly greater proportion of the hatchling S. odoratus and juvenile K. scorpiodes that had been treated with estradiol during embryogenesis than in the untreated group. Results further suggest that estradiol-induced sex reversal seen in TSD turtles is both permanent and ubiquitous and may shed light on the evolution of sex-determining mechanisms in vertebrates.