Peltigera lichens can colonize extreme habitats, such as high-elevation ecosystems, but their biodiversity is still largely unknown in these environments, especially in the southern hemi- sphere. We assessed the genetic diversity of mycobionts and cyanobionts of 60 Peltigera lichens collected in three high Andean steppes of southern Chile using LSU, β-tubulin, COR3 and ITS loci for mycobionts, and SSU and rbcLX loci for cyanobionts. We obtained 240 sequences for the different mycobiont markers and 118 for the cyanobiont markers, including the first report of β-tubulin sequences of P. patagonica through modifying a previously designed primer. Phylogenetic analyses, ITS scrutiny and variability of haplotypes were used to compare the sequences with those previously reported. We found seven mycobiont species and eleven cyanobiont haplotypes, including considerable novel symbionts. This was reflected by ~30% of mycobionts and ~20% of cyanobionts haplotypes that yielded less than 99% BLASTn sequence identity, 15 new sequences of the ITS1-HR, and a putative new Peltigera species associated with 3 Nostoc haplotypes not previously reported. Our results suggest that high Andean steppe ecosystems are habitats of unknown or little-explored lichen species and thus valuable environments to enhance our understanding of global Peltigera biodiversity.