The timing of reproduction is often governed by environmental variables, such as temperature or rainfall. Understanding how environmental variables affect mating dynamics is necessary to predict how systems and populations may adapt to changing environmental conditions and is crucial for management of threatened species. The American horseshoe crab (Limulus polyphemus) ranges from the Yucatan to Maine in distinct populations that differ in their timing of reproduction; while most populations have only one breeding period during the spring, some southern populations have two breeding periods. Here we discuss seasonal patterns of reproduction in a Florida Gulf coast population where horseshoe crabs have two periods of breeding: one in the spring and another in the fall. We used environmental measurements, spawning surveys, mark-recapture, and measurements of adult traits and spawning behavior to compare reproductive parameters between the two spawning seasons over three years. We then evaluated whether environmental conditions affect fall and spring horseshoe crab nesting patterns similarly and whether fall and spring horseshoe crabs should be considered two separate populations. We found significant differences in environmental conditions across seasons and in a wide variety of horseshoe crab traits and nesting parameters. Furthermore, environmental conditions affected nesting behaviors of fall and spring horseshoe crabs differently. However, some individuals spawn during both seasons, suggesting that trait differences may be attributable to environmental effects during development or seasonal plasticity, rather than genetic differences, although further study is necessary. Finally, our results suggest that management practices should be tailored to each population, because environmental conditions may have different effects even on genetically similar groups.