Background: The pes anserine bursa lies beneath the pes anserine tendon, which is the insertional tendon of the sartorius, gracilis, and semitendinosus muscles on the medial side of the tibia, but it can lie in different sites in the medial knee. Accurate diagnosis of the position of the bursa is critical for diagnostic and therapeutic goals. The aim of this study was to evaluate sonoanatomic variations of the pes anserine bursa in the medial knee. Methods: One hundred seventy asymptomatic volunteers were enrolled in this study. Using ultrasound imaging (transverse approach, 7-13 MHz linear array probe) the sonoanatomic position of the pes anserine bursa and its relation to the pes anserine tendon were evaluated. Additionally, we evaluated the sonoanatomic variation of the saphenous nerve. Results: The position of the pes anserine bursa was between the medial collateral ligament and the pes anserine tendons in 21.2%/18.8% (males/females) of subjects; between the pes anserine tendons and the tibia in 67.1%/64.7% (m/f); and among the pes anserine tendons in 8.2%/12.9% (m/f). No significant differences in the position of the bursa existed between males and females. The saphenous nerve was found within the pes anserine tendons in 77.6%/74.1% (m/f) of subjects, but outside the pes anserine tendons in 18.8%/15.3% (m/f). Visibility of sonoanatomic structures was not related to either gender or BMI. Conclusions: Ultrasound provides very accurate information about variations in the pes anserine bursa and the saphenous nerve. This suggests that our proposed ultrasound method can be a reliable guide to facilitate approaches to the medial knee for diagnostic and therapeutic objectives.