Molecular biological techniques now provide many opportunities for determining relationships between organisms. In lichen-forming fungi, polymerase chain reaction (PCR) amplification can be undertaken with primers that have enhanced specificity for fungi, allowing the recovery of precise regions of the genome of the fungal partner. Such PCR techniques are suitable for the analysis of both fresh and dried specimens of great age, and as the techniques require only minimal amounts of starting material, they are suitable for the analysis of large numbers of specimens. PCR can be used to examine both conserved and variable regions of the fungal genome that may be taxonomically significant, and variability in these sequences can be used to describe species boundaries. The ease of analysis of large numbers of specimens without the need to purify or culture the fungal partner makes analysis and comparison of infra- and inter-specific relationships possible, and provides valuable information into their genetic background. Most molecular analyses undertaken with lichen-forming fungi have considered variation within ribosomal RNA clusters, and in this paper we consider the application and interpretation of such PCR-based analysis in the circumscription of species and in determining concepts for individuality and infra-specific groupings.