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In a detailed investigation of lichen diversity in different regions of Pakistan, utilizing molecular and morphological techniques, we identified and reported three species – Dimelaena altissima, Lecanora chlarotera, and Usnea lapponica – as new records for Pakistan. Furthermore, nrITS sequences for Evernia prunastri, Lecanora horiza, L. hybocarpa, Peltigera polydactylon, and P. praetextata were generated for the first time from Pakistan. Detailed morpho-anatomical descriptions, habitat information, distribution data, and results of biochemical analyses are also provided for a better understanding of the lichen diversity and ecology in this region.