The storage of sperm by females across successive reproductive cycles is well documented in internal fertilizers, yet the fate of stored sperm when they compete with ‘new’ sperm to fertilize a female's eggs has rarely been considered. This gap in our understanding is likely due to the logistical difficulties of controlling behavioural interactions during or after mating, which in turn may influence how many sperm are inseminated and how stored sperm are ultimately used during successive bouts of sperm competition with freshly inseminated sperm. Here, we use artificial insemination (AI) in guppies (Poecilia reticulata), a polyandrous live-bearing poeciliid fish exhibiting prolonged sperm storage by females, to overcome these challenges. The use of AI enables us to control potential differential maternal effects (e.g. behaviourally mediated cryptic female choice) and specifically test for post-copulatory paternity biases that favour either stored or fresh sperm when they compete to fertilize eggs. Our paternity analyses revealed the almost complete dominance of freshly inseminated sperm over stored sperm, supporting previous studies reporting similar patterns following natural matings across successive brood cycles. However, our use of AI, which excluded behavioural interactions between males and females, most likely generated a far stronger pattern of fresh sperm precedence compared with those reported in previous studies, possibly implicating ‘cryptic' forms of selection by females that may sometimes bolster the success of stored sperm.