Because of the historical focus of limnology on pelagic processes, the factors controlling lake periphyton growth and nutrient limitation are understudied compared to the phytoplankton. We deployed nutrient‐diffusing substrata at 28 sites spanning a wide trophic status gradient in Lakes Superior and Michigan to assess periphyton biomass accrual on control substrata and the response of periphyton to single and combined phosphorus (P) and nitrogen (N) additions. Periphyton growth was unimodally related to a composite metric of site trophic status, with highest biomass at mesotrophic sites and lower growth at oligotrophic and highly eutrophic sites. Contrary to expectations, P limitation was rare. Instead, several lines of evidence pointed to primary N or N + P co‐limitation of periphyton. Limitation extent was negatively related to site trophic status, with stronger nutrient limitation at oligotrophic sites. Our results support the hypothesis that phytoplankton and periphyton biomass respond differently to nutrient enrichment and suggest that different nutrients may limit pelagic and benthic primary production, even in the same system. Our findings also support the use of periphyton as an early warning indicator of nutrient pollution and help explain why large, oligotrophic lakes may be especially susceptible to localised benthic algal blooms.