Increased strain of the iliotibial band (ITB) is a plausible contributing factor for the development of iliotibial band syndrome (ITBS). Although several studies have found relationships between the strain of the ITB and kinematic factors during running, the associations of the ITB strain with knee alignment and sex, which are considered intrinsic factors, are not well understood. To clarify the sex differences in the ITB strain between genu varum and normal knee alignments in different postures. Observational cross-sectional study. Laboratory research within a university. Forty-four healthy recreational athletes (21 men and 23 women) volunteered for this study and were divided into four groups by sex and knee alignment: men with genu varum alignment, men with normal knee alignment, women with genu varum alignment, and women with normal knee alignment. An ultrasound real-time elastography (RTE) unit was used for distal ITB strain measurements in weight bearing and for different non-weight-bearing: neutral, knee flexion, hip adduction, and hip adduction with knee flexion. Gender information and the intercondylar distance data were collected to divide the participants into two groups. Main Outcome was the ITB strain (strain ratio) measured by the RTE. There were no significant differences in neutral and hip adduction postures among the four groups. However, during weight-bearing, the women's genu varum group (6.91 ± 1.69; Mean ± SD) exhibited greater strain than both the men's normal group (3.50 ± 1.04, P = .005) and the women's normal group (4.42 ± 1.42, P = .048). In addition, there were significant positive correlations between the intercondylar distance and the ITB strain during weight-bearing (r = 0.315, P = .037). The women's genu varum group exhibited a higher ITB strain during weight-bearing, which may be related to the high incidence of ITBS in women athletes. Furthermore, the changes in alignment and muscle activities during weight-bearing could influence the strain of the ITB. III.