Highly variable microsatellite loci were employed to study the mating system of the sexually dimorphic Gulf pipefish Syngnathus scovelli. In this species, like others in the family Syngnathidae, ‘pregnant’ males provide all parental care. Gulf pipefish were collected from one locale in the northern Gulf of Mexico, and internally carried broods of 40 pregnant males were analysed genetically. By comparing multilocus microsatellite fingerprints for the inferred mothers against expected genotypic distributions from the population sample, it was determined that: (i) only one male had received eggs from more than a single female; and (ii) on two separate occasions, two different males had received eggs from the same female. Given the high power to detect multiple matings by males, the first finding indicates that only rarely are individual males impregnated by multiple females during the course of a pregnancy. Conversely, given the lower power to detect multiple matings by females due to sampling constraints, the second finding suggests a high frequency of multiple successful matings by females. Thus, this population of Gulf pipefish displays a polyandrous genetic mating system. The relevance of these genetic findings is discussed with regard to the evolution of secondary sex traits in this species, and in other syngnathids.