﻿AbstractThe fungal mitochondrial small subunit (mtSSU) ribosomal DNA is one of the most commonly used loci for phylogenetic analysis of lichen-forming fungi, but their primer specificity to mycobionts has not been evaluated. The current study aimed to design mycobiont-specificmtSSUprimers and highlights their utility with an example from the saxicolous lichen-forming fungal genusMelaneliaEssl. in Iceland. The study found a 12.5% success rate (3 out of 24 specimens with good-quality mycobiontmtSSUsequences) using universal primers (i.e. mrSSU1 and mrSSU3R), not including off-target amplification of environmental fungi, e.g.CladophialophoracarrioniiandLichenotheliaconvexa. New mycobiont-specific primers (mt-SSU-581-5’ and mt-SSU-1345-3’) were designed by targeting mycobiont-specific nucleotide sites in comparison with environmental fungal sequences, and assessed for mycobiont primer specificity usingin silicoPCR. The new mycobiont-specificmtSSUprimers had a success rate of 91.7% (22 out of 24 specimens with good-quality mycobiontmtSSUsequences) on the studiedMelaneliaspecimens. Additional testing confirmed the specificity and yielded amplicons from 79 specimens of otherParmeliaceaemycobiont lineages. This study highlights the effectiveness of designing mycobiont-specific primers for studies on lichen identification, barcoding and phylogenetics.