A total of six Lasiodiplodia isolates were obtained from acid lime and Valencia orange orchards located in Markaz Bader region. Isolates identification were performed using phylogenetic analysis based on partial sequences of internal transcribed spacer (ITS) sequences, in combination with cultural and microscopic characteristics. Based on ITS sequence data, two species were identified. Only one isolate identified as L. pseudotheobromae and the other five isolates were identified as L. theobromae. All isolates were pathogenic on shoots of acid lime and Valencia orange. Nine ISSR primers were screened with the DNA of the fungal isolates to detect of polymorphism among these isolates. The estimated genetic similarities ranged from 71.7% to 89.1% with Lasiodiplodia isolates. L. pseudotheobromae was previously recorded on mango trees in Egypt, but this is the first time it has been recorded on Egyptian citrus trees.