Background: The Zucchini yellow mosaic virus (ZYMV) is well recognized as a very significant viral pathogen that causes substantial yield reductions in cucurbitaceous crops on a global scale. Methods: To detect and identify ZYMV in squash plants we used the serology reaction, transmission electron microscope, and molecular methods.  Results:  ZYMV was first isolated from naturally infected squash plants in the Alexandria governorate of Egypt, where it caused mosaic, yellowing, and deformation of the leaves. In addition to a molecular diagnosis utilizing RT-PCR, the identification relied on serological responses with antisera to CMV, SqMV, and ZYMV. Using centrifugation and PEG, the virus was cleaned. The A260/280 and A280/260 ratios of the purified virus were 1.38 and 0.72, respectively, which are consistent with those of a nucleoprotein.  2.36 mg of pure virus was recovered from per 100g of infected leaf material. The virus particles, as seen under an electron microscope after a partial purification preparation technique, have a flexuous filamentous structure and measure around 750 nm in length and 12 nm in diameter. Finally, the molecular investigation established the identification of the ZYMV isolate after a specific antiserum with a titer of 1:2.56×10⁴ was generated and tested at the third week.