Biotic and abiotic factors influence the formation of fungal-algal pairings in lichen symbiosis. It is poorly known which factors determine the specific associations, especially when distantly related fungi are considered. Here, we investigated the effect of different drivers on the association patterns of taxonomically diverse lichenized fungi and their trebouxioid symbiotic partners. In 200 samples collected across three biomes, we found 41 species of lichenized fungi, associating with 16 species of trebouxioid green algae, of which 62% were previously unreported. Species identity of the fungal and algal partner had the greatest effect on the outcome of the symbiosis compared with abiotic factors such as climatic variables and geographic distance. Partner specificity was found to be higher in tropical regions than in temperate and arctic regions. Co-phylogenetic analyses indicated congruent phylogenies of trebouxioid algae and associated fungi, rejecting their random associations. Evolutionary mechanisms contributing most to the observed phylogenetic patterns were ‘loss’ and ‘failure to diverge’ of the algal partners. This study broadens our knowledge of fungi-algae symbiotic patterns in view of Trebouxia-associated fungi.