This study explored whether persistent differences in life history traits between populations of Least Killifish Heterandria formosa could be explained by differences in habitat-associated water type, reflecting either local adaptation or phenotypic plasticity. Fish native to a pond and a spring site were reared in the lab in either pond or spring water. Fish reared in pond water reached maturity faster and at the same size as fish reared in spring water, regardless of source population. There was no difference in daily mortality rate between fish raised in the different water types, but the longer time to maturity in spring water produced a lower cumulative survival to maturity. Water type did not affect fecundity, but fish from the spring population had larger offspring, contrary to patterns found in both the field and previously in the lab. Persistent differences among natural populations in life history traits have previously been attributed to differences in population density and predation pressure. While water type affects the expression of some life history traits in Least Killifish, those effects cannot explain the patterns of trait variation among natural populations.