Although application of microsatellite markers to shark biology is in its infancy, these markers are already proving useful for elucidating relationships at population and individual levels. Herein, we provide a preliminary evaluation of microsatellite loci to investigate life history traits and population structure of the bonnethead shark, Sphyrna tiburo. Mature female bonnetheads are known to store sperm in their oviducal gland for a period of about five months after insemination. Although there are no published observations of bonnethead sharks mating, recent studies on other sharks suggest that multiple males often participate in mating events involving a single female. If the bonnethead shark has evolved this type of multiple-male (polyandrous) mating system coupled with protracted sperm storage, it is possible that females are fertilized by multiple males in a single reproductive cycle. Using 8 microsatellite markers, we have assayed several litters of this species for multiple paternity. Three of the 8 microsatellite loci were isolated from the genome of S. tiburo. The remaining five were previously isolated from the blue shark, Prionace glauca, and were also found to be informative for S. tiburo. We also report on a preliminary assessment of micro-geographic population structure in this species from the SE United States.