Microbial nitrification is a critical process governing nitrogen availability in aquatic systems. Freshwater nitrifiers have received little attention, leaving many unanswered questions about their taxonomic distribution, functional potential, and ecological interactions. Here, we reconstructed genomes to infer the metabolism and ecology of free-living picoplanktonic nitrifiers across the Laurentian Great Lakes, a connected series of five of Earth’s largest lakes. Surprisingly, ammonia-oxidizing bacteria (AOB) related to Nitrosospira dominated over ammonia-oxidizing archaea (AOA) at nearly all stations, with distinct ecotypes prevailing in the transparent, oligotrophic upper lakes compared to Lakes Erie and Ontario. Unexpectedly, one ecotype of Nitrosospira encodes proteorhodopsin, which could enhance survival under conditions where ammonia oxidation is inhibited or substrate limited. Nitrite-oxidizing bacteria (NOB) “Candidatus Nitrotoga” and Nitrospira fluctuated in dominance, with the latter prevailing in deeper, less-productive basins. Genome reconstructions reveal highly reduced genomes and features consistent with genome streamlining, along with diverse adaptations to sunlight and oxidative stress and widespread capacity for organic nitrogen use. Our findings expand the known functional diversity of nitrifiers and establish their ecological genomics in large lake ecosystems. By elucidating links between microbial biodiversity and biogeochemical cycling, our work also informs ecosystem models of the Laurentian Great Lakes, a critical freshwater resource experiencing rapid environmental change. IMPORTANCE Microorganisms play critical roles in Earth’s nitrogen cycle. In lakes, microorganisms called nitrifiers derive energy from reduced nitrogen compounds. In doing so, they transform nitrogen into a form that can ultimately be lost to the atmosphere by a process called denitrification, which helps mitigate nitrogen pollution from fertilizer runoff and sewage. Despite their importance, freshwater nitrifiers are virtually unexplored. To understand their diversity and function, we reconstructed genomes of freshwater nitrifiers across some of Earth’s largest freshwater lakes, the Laurentian Great Lakes. We discovered several new species of nitrifiers specialized for clear low-nutrient waters and distinct species in comparatively turbid Lake Erie. Surprisingly, one species may be able to harness light energy by using a protein called proteorhodopsin, despite the fact that nitrifiers typically live in deep dark water. Our work reveals the unique biodiversity of the Great Lakes and fills key gaps in our knowledge of an important microbial group, the nitrifiers.