Zucchini yellow mosaic virus (ZYMV) was isolated from naturally infected squash plants exhibiting systemic vein-banding, yellowing, mosaic, leaf deformation and stunting symptoms. In this study, the virus isolate was identified using biological, serological and molecular techniques as ZYMV. In biological analysis, the isolate produced severe symptoms on susceptible cucurbit hosts and local lesions on leaves of indicator plants. Direct enzyme linked immunesorbent assay and direct tissue blot immunosorbent assay (DTBIA) were successfully used to detect ZYMV isolate in squash plants. Electron microscopy of leaf dip preparation of infected squash leaves showed long flexuous filamentous virus particles of size (750X13 nm). Reverse transcription polymerase chain reaction (RT-PCR) was carried out using ZYMV-specific primers, designed to amplify a 1005-bp fragment covering the entire coat protein (CP) gene and part of 3'-untranslated region (3′-UTR) from infected squash plants. The partial nucleotide sequence of the CP gene of ZYMV-EG isolate has been deposited in NCBI GenBank under accession number KU127244. According to the sequence analysis the Egyptian isolate has specific amino acids sequences that are different from other ZYMV isolates. Comparison with 30 ZYMV sequences retrieved from GenBank presented nucleotide identities in the range of 95-97%, amino acids sequences similarities ranged from 92.23-96.12%. Phylogenetic analysis revealed that ZYMV-EG isolate was grouped into a distinct clade comprising Taiwan isolates (TW-TN3 and TW-NT1), Chinese isolate (Hangzhou) and Brazilian isolate (ZYMV-DF) in the cluster A which is apparently the most widespread throughout the world.