Interaction patterns between lichen-forming fungi and their photobionts vary across spatial and evolutionary scales. The eco-evolutionary mechanisms driving interaction turnover are not fully understood for lichens, mainly because thorough ecological sampling at multiple nested spatial scales, including at an inter-biome scale, has not been implemented. A recent study of the lichen genus Peltigera and its Nostoc cyanobionts at an intra-biome (boreal) scale revealed that bioclimatic factors are more limiting than cyanobiont availability for explaining geographic distributions of Peltigera species. However, at the spatial and bioclimatic scale of that study, two main generalist Nostoc phylogroups dominated across the boreal biome. Therefore, an understanding of the spatial scale and drivers of Nostoc distribution, as well as the effect of environmental gradients and stochasticity on the occurrence of different symbiotic pairs, is still lacking. We are using the large-scale, systematic, multi-biome sampling of Peltigera specimens of the Alberta Biodiversity Monitoring Institute to ask three main questions: (1) What is the spatial scale, and associated environmental factors, of Nostoc phylogroup replacement? (2) How does this shape the structure of Peltigera–Nostoc interaction networks? (3) How is this constrained by their evolutionary relationships? We expect that the replacement of dominant Nostoc phylogroups occurs mostly at an inter-biome scale and is correlated with environmental factors that vary across the six biomes included in our sampling. We also predict that this inter-biome turnover will be reflected in the overall network structure, where modules will be restricted to specific biomes and there will be one main generalist (central) Nostoc phylogroup per module. Our results highlight the complex interplay between these eco-evolutionary drivers of interaction turnover at different spatial scales, shedding light on the assembly process of lichen communities.