Symbiosis is a major theme in the history of life and can be an important force driving evolution. However, across symbioses, it is difficult to tease apart the mechanisms that structure the interactions among potential partners. We used genetic similarity and frequency-based methods to qualitatively and quantitatively examine the patterns of association among several co-occurring Cladonia lichen fungi and their algal photobionts in six disjunct Florida scrub sites. The patterns of association were described by the degree of specificity, i.e. the phylogenetic range of associated partners, and of selectivity, i.e. the frequency of association among partners. Six fungal species associated with only one algal internal transcribed spacer clade, with the remaining two fungi being associated with two algal clades. In all cases, the fungi associated in unequal frequencies with the observed algal photobiont genotypes within those clades — suggesting that both specificity and selectivity were higher than expected. Fungal species can be grouped into three significantly different specificity classes: photobiont specialists, intermediates and generalists. In contrast to the pronounced specificity for photobionts among fungal species, the different Florida scrub sites do not harbour distinct photobiont pools, and differential photobiont availability cannot explain the patterning of lichen associations at this spatial scale. Therefore, we conclude that fungal specificity and selectivity for algal photobionts are major factors in determining the local composition of symbiotic partnerships.