<p>Understanding the nerve distribution within the vastus lateralis muscle in relation to anatomical landmarks is essential for effective botulinum neurotoxin injections to manage chronic anterior knee pain and vastus lateralis muscle tone disorders. This study proposes an anatomically informed approach for administering these injections to the vastus lateralis muscle. Using a modified Sihler’s method, we examined nerve distribution in the vastus lateralis muscles of 12 fresh cadavers. The muscles were analyzed with respect to a transverse line crossing the greater trochanter of the femur and the base of the patella, dividing them into four zones from top to bottom. Ultrasonography was used to examine the muscle anatomy and to guide injections, confirming both structural details and injection accuracy. Intramuscular nerve distribution in the vastus lateralis muscle showed significant patterns, particularly in zones 2 and 3. Based on the nerve distribution and surface landmarks, ultrasonography-guided injections showed high accuracy without damage to surrounding essential anatomical structures. Targeting botulinum neurotoxin injections at the areas with the densest nerve distribution is recommended. Adhering to these guidelines enables clinicians to use minimal doses and reduce the risk of adverse effects such as gait disturbances, antibody production, and bruising from multiple injections.</p>