An emerging and expanding disease of date palm orchids at El-Kharga and El-Dakhla Oases at the New Valley Governorate in Egypt was observed since 2007. The leaves of diseased plants were hanged to the trunk with rotting of the leaf base part. Rotting of leaf base was started partially at one side of the leaf base, then the leaf base was completely rotted causing a weakness leaf which was separated partially and hanged to the trunk. The causal pathogen was isolated from rotted leaf base of date palm and subsequently identified as Chalara state of Ceratocystis radicicola (Bliss) C. Moreau. It is worthy to mention that this is the first time to isolate Ceratocystis radicicola in New Valley. However, this fungus was recovered from approximately 100% of different locations and scored 50.2% of frequency among all isolated fungi. On the other hand, the other isolated fungi appeared in low frequency. Seven representative isolates of C. radicicola, were selected for Inter Simple Sequence Repeat (ISSR) analysis. These isolates represented different morphological variants and different localities in El-Kharga and El-Dakhla Oases, New Valley, Egypt. Based on ISSR marker polymorphisms, results verify that the most distant relationship was scored between the two isolates, Cr. 5 from site 5at El-Kharga Oases and Cr. 7 isolated from site 7 at El-Dakhla Oases. The dendrogram of the seven isolates cluster comprised three isolates, Cr. 5isolated from site 7 at El- Kharga Oases, Cr. 6 isolated from site 6 at El-Kharga Oases and Cr. 4 isolated from site 4 at El-Dakhla Oases which were closer in their relationship. the results highlight that attention must be given to the importance of C. radicicola, since it was isolated from all fields and scored the highest values on the fungal frequency at all locations of El-Dakhla and El-Kharga Oases.