Introduction: The anterolateral thigh flap is an example of a free flap widely applied for reconstruction of tissues within various areas of the body. Aim: The aim of the study was to determine the most common locations of the ALT flap perforators in men and women as well as to clarify any potential differences in the location of the vessel in male vs. female subjects. Material and method: In years 2021–2022, a study was conducted at the Plastic, Reconstructive, and Aesthetic Surgery Clinic in a group of 90 volunteers (45 women and 45 men, a total of 180 lower extremities) to determine the location of the anterolateral thigh flap perforator against the flap surface using a portable Doppler probe. Results: Variable locations were revealed for the anterolateral thigh flap perforator within the study. In women, the perforator is located more distally from the anterior superior iliac spine, most frequently within the inferolateral quadrant, while being located closer to the anterior superior iliac spine, and most frequently within the superolateral quadrant, in men. No perforator was found in 3.3% of the examined limbs. No statistically significant differences were observed between the course of the perforators within the left and right lower limbs in individual patients.Conclusions: The location of the ALT flap perforator is subject to sexual dimorphism, which facilitates the investigation area being narrowed down to the well-defined locations of its most frequent occurrence when planning anterolateral thigh flap reconstructive procedures.