In this study, the focus was on the bioinformatics analysis of the coat protein (cp) gene of a specific Egyptian isolate of Zucchini yellow mosaic virus (ZYMV), which was identified based on both biological and molecular characteristics. The presence of ZYMV was confirmed in 27 squash samples using DAS-ELISA and RT-PCR techniques employing ZYU-F and ZYD1186-R primers that flank the cp gene of ZYMV. Transmission experiments involving mechanical and aphid transmission, specifically through Aphis gossypii, were carried out on various hosts, including Chenopodium amaranticolor, which served as an indicator necrotic local lesion host. Using light and electron microscopies, amorphous and cylindrical inclusions (pinwheels and scrolls) were observed, respectively. A partially purified virus preparation of ZYMV was obtained from infected squash leaves using a protocol involving polyethylene glycol purification and ultracentrifugation, revealing the presence of filamentous viral particles that were negatively stained with 2% uranyl acetate. Subsequently, the ZYMV-cp gene was amplified via RT-PCR for sequencing following the purification of PCR products from the agarose gel. The nucleotide sequences obtained, consisting of 831 base pairs, and encoding a 236 amino acid open reading frame, were deposited under the ID: LC778450.1 as the ZYMV-Shrouk-23 strain in GenBank. Bioinformatics analyses were conducted, comparing the Egyptian isolate to 25 overseas isolates of ZYMV. The resulting phylogenetic analysis dendrogram revealed a distinct clade with identities ranging from 91.94% to 99.16% based on nucleotide sequences and from 96.19% to 100.00% based on deduced amino acid sequences.