The Mediterranean widow spider, or "Dolmak," Latrodectus tredecimguttatus (Rossi, 1790), family Theridiidae, is one of the medically significant spiders. Also, it is listed as a dangerous animal due to its extensive geographic range and its deadly or seriously problematic toxic bites. A holistic understanding of L. tredecimguttatus requires combining knowledge derived from both morphological and molecular investigations. Thus, this study aimed to offer researchers a framework for investigating these essential species and to educate farms and visitors about the importance of avoiding any behaviour that could antagonize the spiders. For this study, specimens of L. tredecimguttatus were manually collected randomly from some olive orchards between September 2023 and August 2024 at Sadat City, Menoufia Governorate, Egypt. The morphological parameters utilized for identification were the total body length, carapace width, abdomen length, and (1^st,3^rd, and 4^th legs) length. Results indicated that the total length of females ranged from 8.92 to 10.15mm. The female dorsal pattern if present; consists of several orange spots (usually in young females). Adult females lack the dorsal pattern and ventral hourglass reduced to two transverse oranges to red Markings. The results of the mitochondrial CO1 gene confirm that our tested species identified as Latrodectus tredecimguttatus under the accession number (PQ358290.1) in GenBank for the first time in Menoufia Governorate, Egypt. So, the identification of a female Black Widow according to morphological and molecular genetic methods is of great importance.