Timing of maturation is an important life-history trait that is likely to be subjected to strong natural selection. Although population differences in timing of maturation have been frequently reported in studies of wild animal populations, little is known about the genetic basis of this differentiation. Here, we investigated population and sex differences in timing of maturation within and between two nine-spined stickleback (Pungitius pungitius) populations in a laboratory breeding experiment. We found that fish from the high-predation marine population matured earlier than fish from the low-predation pond population and males matured earlier than females. Timing of maturation in both reciprocal hybrid crosses between the two populations was similar to that in the marine population, suggesting that early timing of maturation is a dominant trait, whereas delayed timing of maturation in the pond is a recessive trait. Thus, the observed population divergence is suggestive of strong natural selection against early maturation in the piscine-predator-free pond population.