Zucchini yellow mosaic virus (ZYMV) is one of the most destructive viruses which cause significant economic losses in many cucurbits crops in Egypt. The purpose of this study was to use biological and molecular techniques to characterize zucchini yellow mosaic virus (ZYMV) stain isolated from open fields of Sharqia Governorate. ZYMV was isolated from infected zucchini plants showing virus like symptoms, samples were collected from open fields of Sharqia Governorate In different seasons (2019-2020). The fruit samples of zucchini exhibited severe mosaic, blisters, yellowing and malformation. The isolated virus was detected biologically to different species belonging six families as (Cucurbitaceae, Chenopodiaceae, Leguminoseae, Graminaceae, Solanaceae and Asteracea) by mechanical inoculation, serologically by DAS-ELISA (Double antibody sandwich enzyme linked immunesorbent assay) and also the stability of ZYMV was studied, found that Thermal inactivation point (TIP), Dilution end point (DEP) and Longevity in vitro (LIV)] were 65ºC, 10-4, and 96 hours, respectively. Purified virus sample was negatively stained and examined under electron microscopy (EM) revealed flexuous filamentous particles approximately (680-840) nm long and (12-14) nm in diameter. ZYMV was molecularly identified by the reverse transcription polymerase chain reaction (RT-PCR), the size of the potyvirus P1 gene was shown to be about 600 bp using sodium dodecyl sulphate–polyacrylamide gel electrophoresis. ZYMV P1 gene was quite similar, with a nucleotide sequence similarity of 98 % between several ZYMV previously isolated.