Objective Gender is a risk factor in the onset of osteoarthritis (OA). The aim of the study was to investigate gender differences in contact area (CA) and congruity index (CI) in the medial tibiofemoral (MTF) joint in 2 different cohorts, quantified automatically from magnetic resonance imaging (MRI). Design The CA and CI markers were validated on 2 different data sets from Center for Clinical and Basic Research (CCBR) and Osteoarthritis Initiative (OAI). The CCBR cohort consisted of 159 subjects and the OAI subcohort consisted of 1,436 subjects. From the MTF joint, the contact area was located and quantified using Euclidean distance transform. Furthermore, the CI was quantified over the contact area by assessing agreement of the first- and second-order general surface features. Then, the gender differences between CA and CI values were evaluated at different stages of radiographic OA. Results Female CAs were significantly higher than male CAs after normalization, male CIs were significantly higher than female CIs after correcting with age and body mass index ( P < 0.05), consistent across the 2 data sets. For the OAI data set, the gender differences were present at all stages of radiographic OA. Conclusion This study demonstrated the gender differences in CA and CI in MTF joints. The higher normalized CA and lower CI values in female knees may be linked with the increased risk of incidence of radiographic OA in females. These differences may help further understand the gender differences and/or to establish gender specific treatment strategies.