Environmental and genetic factors influence fish spawning periodicity (i.e., the distribution of spawning events during the breeding season), but their relative contributions have rarely been evaluated. We evaluated the relative contribution of genetic and environmental effects on spawning periodicity by rearing Florida largemouth bass (FLMB, Micropterus salmoides floridanus) from Lake Okeechobee and intergrade largemouth bass (ILMB, Micropterus salmoides salmoides × M. s. floridanus) from Lake Seminole in a similar environment. Fish from each genetic source population were translocated to experimental ponds at an intermediate latitude in Gainesville, Florida, in September 2003. We used estimated ages of offspring as an index of spawning events to compare spawning distributions between brood sources in ponds and related those results to spawning distributions at source populations for 2004. FLMB began spawning earlier than ILMB in all ponds, and FLMB had a longer spawning season than ILMB. Similarly, FLMB at Lake Okeechobee began spawning earlier and had a longer spawning season than ILMB at Lake Seminole. Environmental factors (e.g., temperature effects) influenced spawning periodicity for both FLMB and ILMB, but spawning periodicity was also influenced by genetic composition in ponds because translocated fish reflected characteristics of their source populations. Thus, both environmental factors and genetic composition influenced spawning periodicity.