Asexual reproduction via parthenogenesis is relatively rare among chordates and has never been recorded in the class Chondrichthyes. In December 2001, a female Bonnethead Shark, Sphyrna tiburo, gave birth in captivity to a single female pup, despite having been separated from any male S. tiburo for a period of at least three years. Widespread media attention quickly led to this case being billed as "the shark-virgin birth" (i.e., asexual reproduction), however other explanations (sexual reproduction coupled with long-term sperm storage, inter-specific hybridization with a male leopard shark, Triakis semifasciata, tank-mate) could not be ruled out. We present the results of a genetic investigation aimed at ruling out these alternatives and determining whether this birth is the first known case of asexual reproduction in this ancient lineage.