Abstract Introduction The influence of limb malrotation on long-leg radiographs (LLR) is frequently discussed in literature. This systematic review aimed to describe the influence of limb rotation on alignment measurements alone and in combination with knee flexion, and determine its clinical impact. Materials and methods A literature search was conducted in June 2021 using the databases MEDLINE, Cochrane, Web of Science (Clarivate Analytics), and Embase. The search term ((radiograph OR X-ray) AND (position OR rotation) AND limb alignment) was used. Database query, record screening, and study inclusion and exclusion were performed by two reviewers independently. Experimental studies (using either specimens or synthetic bones) or clinical studies (prospective or retrospective using radiographs of patients) analyzing the influence of limb rotation on anatomic and mechanical limb alignment measurements were included. Characteristics and results of the included studies were summarized, simplified, and grouped for comparison to answer the research question. Studies were compared descriptively, and no meta-analysis was performed. Results A total of 22 studies were included showing large heterogeneity, comprising studies with cadavers, patients, and synthetic bones. Most studies (7 out of 8) reported that external rotation (ER) causes less apparent valgus and leads to more varus and internal rotation (IR) causes more valgus and leads to less varus. However, there is no consensus on the extent of rotation influencing alignment measures. Studies reported about an average change of > 2° (n = 4) and < 2° (n = 4) hip-knee-ankle angle (HKA) between 15°IR and 15°ER. There is a consensus that the impact of rotation on mechanical alignment is higher if additional sagittal knee angulation, such as knee flexion, is present. All five studies analyzing the influence of rotation combined with knee flexion (5°–15°) showed an HKA change of > 2° between 15°IR and 15°ER. Conclusion Malrotation is frequently present on LLR, possibly influencing the measured alignment especially in knees with extension deficit. Surgeons must consider this when measuring and treating deformities (high tibial osteotomy or total knee arthroplasties), and analyzing surgical outcomes. Especially in patients with osteoarthritis with knee extension deficits or postoperative swelling, the effect of malrotation is significantly greater.