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Coffee leaf rust (CLR) is the most devastating disease in coffee (coffea sp.) and caused by the rust fungus Hemileia vastatrix. Currently, there are several studies on developing disease resistance varieties. Thus, long-term live storage of H. vastatrix is necessary. Hemileia vastatrix is a biotroph pathogen, which requires nutrients from living host cells and cannot be cultivated in vitro. However, to continue culturing CLR in living plants is not only labor intensive, but also not cost effective. Therefore, three cryopreservation techniques and two storage temperatures were investigated on three potential CLR isolates – DC204, DC404 and DC604 – collected from Chiang Rai province, Thailand. Although, all cryopreservation techniques failed to provide long term storage of H. vastatrix, in situ cryopreservation at -20 oC showed potential for short term storage of this pathogen. Thus, this technique could be used as an alternative to in vivo culturing for maintaining pathogenic isolates in the laboratory.