In many animal species, members of one sex, most often females, exhibit a strong preference for mating partners with particular traits or resources. However, when females sequentially mate with multiple partners, strategies underlying female choice are not very well understood. Particularly, little is known if under such sequential polyandry females mate truly randomly, or if they actively try to spread mating events across multiple partners. In the present study, we used the highly promiscuous poison frog to investigate whether promiscuity could result from a preference for novel mates. Furthermore, we examined the importance of call characteristics for mate choice. We conducted mate choice experiments in a laboratory setup, by presenting females with recent mating partners or novel males. We recorded call characteristics of both males and the time females spent close to each male. In our trials, females preferred previous mating partners over novel males and also males with shorter advertisement calls. Results from previous studies on suggest that females in our trials recognized previous partners based on individual call characteristics. While mating decisions in the wild and in the laboratory might differ, our study provides first evidence for female mate choice in a poison frog with sequential polyandry.