Euphorbia peplus (Family Euphorbiaceae) is a medicinal annual weed found in many habitats in Egypt. The main goal of this study was to assess the diversity of E. peplus accessions growing in Egypt and to measure the levels of genetic characters of six selected accessions collected from different habitats in Egypt, by using Inter-Simple Sequence Repeat (ISSR), SDS-PAGE, and isozymes. Extracting, measuring, and identifying the bioactive compounds were also taken in our consider. A total of 45 plant species were recorded as associates with E. peplus in various habitats. Poaceae and Asteraceae were the most common plant families. Therophytes were the most abundant life forms. Cosmopolitan species were the most common floristic category. Cynodon dactylon, E. peplus, and Sisymbrium irio had the highest importance values, of 58.3, 47.6, and 46.8, respectively. A total of six primers were evaluated for ISSR–PCR analysis, and the polymorphism percentages varied from 20–66.66%. Primer HB-9 produced the maximum number of polymorphic products (four bands), while primer HB-12 produced the lowest number of polymorphic products (one band). SDS-PAGE analysis revealed some variations among the studied accessions in the banding pattern and 50 % polymorphism. Two isozymes (peroxidase and polyphenol oxidase) were used to distinguish among the six accessions of E. peplus. The obtained data showed genetic and phytochemical compounds content variation among the six studied samples.