Physiological characteristics of gametogenesis, fertilization, and early larval development in Ostrea chilensis (Philippi, 1845) pose a number of challenges for selective breeding, despite strong commercial potential. In wild populations, this larviparous protandric hermaphrodite exhibits asynchronous gonadal maturation and relatively low fecundity. Reproductive success and genetic diversity in a hatchery population are primarily determined by female fecundity and fertility, as well as synchrony of female gonad development. Better hatchery control of the reproductive cycle can lead to more cost-effective and reliable breeding. This study examined factors such as feed and temperature in an attempt to increase reproductive rates and female sex ratios in brood stock. Oysters held under two different hatchery conditioning regimes—flow-through outdoor nursery ponds and temperature-controlled indoor tanks—spawned earlier and had higher reproductive rates than natural or farmed populations. Oysters were sampled over 6 months with histological analysis used to assess seasonal gamete patterns. Magnetic resonance imaging was also trialed and compared with histology findings. Significant increase in female gonad proportion and improved synchrony of egg maturation was observed through manipulation of feed and temperature. The implications of these findings for implementing a cost-effective selective breeding program in this species are outlined.