Guava die-back disease is a destructive disease. It causes serious yield losses and reduced guava fruits. Inspection of guava plant for die-back revealed that Beheira governorate had the highest frequencies of all isolated fungi from natural infected guava trees, followed by Alexandria, while Kafr El-Sheikh obtained the lowest frequency. Botryodiplodia theobromae gave the highest frequency of isolated fungi from natural infected guava trees during seasons 2015 and 2016, followed by Alternaria alternata, Alternaria sp., whereas Colletotrichum gloeosperiodes, Phomopsis sp., and Nigrospora sp. obtained minor frequency. The highest frequency of Botryodiplodia theobromae was observed at Beheira, followed by Alexandria governorate. Meanwhile, the lowest frequency was found at Kafr El-Sheikh. Pathogenicity test indicated that Botryodiplodia theobromae was the most pathogenic fungus. RAPD analysis of DNA by using three random primers exhibited five groups of DNA bands with first primer, six groups with second primer and five groups with the third primer and the overall similarities between isolates were arranged from 69 to 100% with P1, from 62 to 100% with P2 and from 33 to 100% with P3. The present study aimed to finding correlation between these isolates groups which resulted of RAPD, its pathogenesis and its geographical isolated locations. Ten isolates of B. theobromae represented ten locations of five governorates in Egypt were tested to evaluate the ability of pathogenesis in guava transplants cultivar Banaty at pots under experiments greenhouse conditions for artificial inoculation. The obtained results were exhibited that all tested isolates were clearly pathogenic to guava transplants and produced typical symptoms of guava die-back disease. Such isolates were differences of differed in their pathogenicity. The  ten isolates were coded according to their locations by three random primers (B.W.1, A.B.2, D.S.3, Q.K.4, A.A.5, B.N.6, K.Q.7, Q.S.8, D.B.9 and K.D.10). Isolate code No. Q.K.4 was the most virulent, followed by D.S.3 and Q.S.8, respectively which isolated of Qalyoubia and Damietta governorates. Meanwhile isolates K.D.10, A.A.5, K.Q.7 were moderately pathogenic, they were isolated from Kafr El-Sheikh and Alexandria, while A.B.2 and B.N.6 were the lowest pathogenic isolates isolated from Alexandria and Beheira governorates.