This study aimed to identify and characterize the most common Anisakis larvae infecting Atlantic horse Mackerel (Trachurus trachurus) in the South Mediterranean basin; based on morphological and advanced molecular characterization. Also, the linkage between the prevalence of Anisakis spp. larvae and some biological variables including Mackerel age and sex were also investigated. Moreover, the clinical intensity of larvae infection in different organs of infected Mackerel was assessed. Atlantic horse mackerel collected from the southern Mediterranean shores were found to be infected with third stages larvae (L3) of Anisakis species. The Anisakis larvae were found encapsulated as coiled tightly in different fish organs and uncoiled freely in the abdominal cavity of infected T. trachurus. These larvae were categorized into 3 types of Anisakis spp. as A. simplex type1; A. pegreffii; Anisakis simplex / pegreffii hybrid based on their morphological and molecular characterizations. Subsequently, the phylogenetic analysis of ITS region of different investigated larvae confirmed the identification of collected Anisakis spp. The remarkably high intensity of Anisakis spp. larvae in Mackerel gonads warns of possible future deleterious impacts on the growth, development and sustainability of Mackerel fisheries at the south Mediterranean coasts.