We used data from a long-term field study of two populations of the Least Killifish, Heterandria formosa, to examine whether genetically based population differences in offspring size at birth are mainly due to differences in pre-fertilization offspring provisioning (i.e., differences in egg mass) or differences in post-fertilization offspring provisioning (i.e., the degree of matrotrophy). We found differences between populations in pre-fertilization offspring provisioning and larger differences in post-fertilization provisioning. These results establish population variation in H. formosa as a potential model for studying the costs and benefits that could modulate the evolution of matrotrophy. In addition, our results illuminate some of the costs and benefits associated with matrotrophy and offer insight into how matrotrophy influences the expression of other life history relationships, including that between female size and fecundity, the trade-off between offspring number and size, and the population-specific responses of offspring number and size to variation in population density.