While intensified nutrient limitation of periphyton has been reported along wetland nutrient-depletion gradients, changes to the specific nutrient that limits periphyton growth are not documented. In this study, we used artificial nutrient-diffusing substrata to determine nutrient limitation status of periphyton along a nitrogen- and phosphorus-depletion gradient in a freshwater marsh during the growing season of 2003. We also characterized water-column nutrient content, N:P ratio of dissolved nutrients, and periphytic N₂ fixation potential along the gradient. Dissolved inorganic nitrogen concentrations consistently decreased (60% − 95%) from inflow to outflow during all bioassays, while soluble reactive phosphorus concentrations decreased during the April (49%) and September (39%) bioassays but increased in the July bioassay (51%). Unequal N and P retention resulted in a general decrease in DIN:SRP mass ratio from 20.2 ± 5.0 to 3.8 ± 1.6 between inflow and outflow, respectively. Periphyton at the wetland inflow never responded to N additions alone, while periphyton at the outflow always responded to N enrichment. Periphyton at the inflow were either not limited by nutrients (September) or co-limited by N P (April and July). At the outflow, periphyton were either N-limited (April and September) or strongly co-limited by N P (July). A significant increase in N₂ fixation potential (p < 0.05) from inflow to outflow locations was noted for all measured events. Our results suggest that, in addition to the severity of nutrient limitation, some wetlands may display spatial heterogeneity in the specific nutrient that limits periphyton growth. Further, these shifts influence the structure and function of wetland periphyton assemblages.