ABSTRACT From May 2016 until December 2018, a group of blacktip reef sharks ( Carcharhinus melanopterus ) at Shedd Aquarium successfully produced six litters. These multiple successful breeding events provided the opportunity to develop and characterize microsatellite loci in this species to properly identify paternity for future studbook succession. Thirteen microsatellite loci were developed to determine the paternal lineages of all six litters born to four female and three male blacktip reef sharks. Of the six litters produced, three were the result of polyandrous mating, including one litter that was sired by all three males. While some females only produced one litter during the study, other females exhibited a biennial reproductive cycle. Although all three males had equal opportunity to mate with various females, one male sired over half of the offspring produced during this 2‐year period. This study highlights the importance of using paternity analysis as a tool to aid in studbook management and future ex situ population conservation decisions.