Intron origin and evolution are of high interest, yet the rates of insertion and loss are unclear. To investigate their spread, we studied ribosomal (r)DNA introns from the closely related lichens Physcia aipolia and P. stellaris. Both taxa are replete with rDNA spliceosomal introns and autocatalytic group I introns, many of which show presence/absence polymorphism when screened with the PCR approach. This initially suggested that Physcia could be a model for studying intron retention and loss. However, during the course of a population-level analysis, we discovered widespread intron presence/absence heterogeneity within lichen thalli. To address this result, we sequenced multiple clones encoding nuclear rDNA and the single-copy elongation factor-1alpha (EF-1alpha) from individual thalli. These data showed extensive rDNA heterogeneity within individuals, rather than the presence of multiple fungi within a thallus. Our results suggest that considerable care must be taken when interpreting intron presence/absence in lichen rDNA, an observation that has general implications for the study of rDNA intron evolution.