Zucchini yellow mosaic virus (ZYMV) is one of the most important viruses and is responsible for significant losses in cucurbit crops worldwide. In Egypt, it causes serious economic losses in squash especially in spring season. During 2014-2015, samples were collected from squash fields in different Provinces, Fayoum Governorate. In Fayoum, ZYMV-naturally infected squash plants exhibited severe mosaic and blisters, leaves deformation, blisters on the fruits and discoloration hardening of flesh, as well as external fruit cracks. Percentages of infection in squash cv. Eskandrani, ranged between 20-35% and 70-78% in mid-March and May, respectively. Meanwhile, it ranged between 15-25% in September and 62% in the end of November. The virus was isolated from naturally-infected squash plants serologically depending on enzyme-linked immunosorbant assay (ELISA), and biologically by mechanical inoculation to different species belonging four families i.e., Amarnthaceae, Chenopodiaceae, Cucurbitaceae, Solanacea. In virus vector relationship, transmission rates recorded 84.6, 86.6, 93.3, 90, and 91.6%, respectively for squash cvs. Alia, Asma, Eskandrani, Mabrouka and Safa. In this context, one insect vector (Myzus persicae) proved its ability to transmit ZYMV. Electron microscopy (EM) of partially purified preparation method revealed flexuous particles approximately 750X15 nm. Serologically, TBIA proved to be effective in detecting ZYMV in the infected samples and reliable technique for the virus detection in squash leaf midrib and petioles. Electrophoresis analysis of the reverse transcription polymerase chain reaction (RT-PCR) amplification revealed that the primers amplified a product size of 458bp for the RNA extracted from ZYMV-infected tissues.