Increased patellofemoral joint (PFJ) stress has been implicated in the development of PFJ pathologies. Previous studies have identified a relationship between reduced hamstring length and patellofemoral pain syndrome. Hamstring stretching is also recommended in the management thereof. However, the relationship between reduced hamstring length and PFJ stress has not been explored in vivo during activities that load the PFJ, such as squatting. The objective of this study was to determine if persons with reduced hamstring length demonstrate increased PFJ stress during squatting compared with individuals without reduced hamstring length. Eight participants with, and eight participants without, reduced hamstring length were assessed to determine their PFJ contact area using magnetic resonance imaging, and their PFJ reaction force during squatting using motion analysis. Data collected were entered into a biomechanical model to calculate medial, lateral and total PFJ stress. It was found that participants with reduced hamstring length had significantly greater total (393.39 Pa/kg vs. 213.01 Pa/kg) and lateral (311.23 Pa/kg vs. 142.55 Pa/kg) PFJ stress at 60° knee flexion during squat descent and ascent (427.75 Pa/kg vs. 255.64 Pa/kg and 337.75 Pa/kg vs. 170.63 Pa/kg, respectively). This was due to significantly increased PFJ reaction force at 60° knee flexion during squat descent (12.18 N/kg vs. 7.21 N/kg) and ascent (13.03 N/kg vs. 8.72 N/kg), and lower medial PFJ contact area at 60° knee flexion (88 mm2 vs. 160 mm2). The results of this study demonstrate a relationship between reduced hamstring length and increased PFJ stress during squatting due to increased PFJ reaction force and reduced medial PFJ contact area. © 2009 Elsevier B.V. All rights reserved.