OBJECTIVES: Selective atrophy of vastus medialis oblique (VMO) may be present in patellofemoral pain (PFP). This study investigated the validity of real-time ultrasound in measuring the thickness of each quadriceps muscle. DESIGN: Cross sectional-Validity. SETTING: University laboratory. PARTICIPANTS: 10 limbs, 5 people with unilateral PFP. MAIN OUTCOME MEASURES: The thickness of VMO, vastus lateralis (VL), vastus intermedius (VI), rectus femoris (RF), and vastus medialis (VM) measured with ultrasound were compared to magnetic resonance imaging (MRI) muscle thickness measurements, using Pearson's (r), and compared to MRI muscle cross-sectional area (CSA) measurements, using Spearman's correlation coefficient (rho). RESULTS: There was a good correlation between ultrasound and MRI measures of the thickness of each superficial quadriceps muscle VMO (r = 0.86), VM (r = 0.86), VL (r = 0.94), RF (r = 0.86), and a poor for VI (r = 0.37). Ultrasound measures had a good correlation to MRI muscle CSA measures for VL (rho = 0.83) and RF (rho = 0.88), moderate for VM (rho = 0.73), and poor for VMO (rho = 0.20), and VI (rho = 0.310). CONCLUSION: Real-time ultrasound muscle thickness measurements are correlated to MRI measured thickness of superficial quadriceps muscles (VMO, VL, VL, and RF) in PFP.