After long periods of storage, plant pathogen isolates lose their sporulation capacity. The objective of this study was to evaluate re-isolation methodologies for recovering sporulation of Pestalotiopsis grandis-urophylla isolates after subjection to a long period of storage. Isolates of P. grandis-urophylla were kept for 14 months on Petri dishes with PDA medium at 10°C. After this period, the isolate colonies showed reduced mycelial growth and no sporulation. The isolates were inoculated on healthy Eucalyptus grandis-urophylla leaves, and after ten days they were subjected to three re‑isolation methods: scraping of the lesions (S) removing of injured plant tissue fragments, followed by disinfestation (D) and without disinfestation (WD). Then, the purified isolates were evaluated for the recovery of its sporulation ability. The different methods for re-isolation resulted in the occurrence of differences among the isolates, showing that sporulation is an isolate-dependent feature. The three methods (S, WD and D) allowed the sporulation recovery of P. grandis-urophylla, even after these isolates have been subjected to 14 months.