The aim of this study was to compare the muscle activity of the gluteus medius (GMe), gluteus maximus (GMa), biceps femoris (BF), vastus lateralis (VL), vastus medialis (VM) and erector spinae (ES) as well as medial knee displacement (MKD) while using varying stiffness resistance bands (red: 1.68 kg; black: 3.31 kg; gold: 6.44 kg) during a barbell back squat (BBS) among males and females. A total of 23 (females: 11) resistance trained people were recruited for this study. Muscle activity was measured using electromyography, and motion capture cameras tracked lower-limb kinematics and MKD. Three resistance bands were placed at the distal end of the femur while performing a BBS at their 85% repetition maximum (RM). Parametric and non-parametric statistical analyses were conducted with the alpha level of 0.05. The gold resistance band resulted in a smaller knee-width-index value (i.e., greater MKD) compared to other bands (p < 0.01). Males exhibited less MKD compared to females during the BBS for each resistance band (p = 0.04). Males produced greater VL activity when using the black and gold resistance bands during the BBS (p = 0.03). When using a gold resistance band, the GMe muscle activation was higher compared to other resistance bands (p < 0.01). VM muscle activity was reduced when using a gold resistance band compared to no band condition (p < 0.01). BF (p = 0.39) and ES (p = 0.88) muscle activity did not change when using different resistance bands. As a result, females may be at a biomechanical disadvantage when using resistance bands compared to males while performing the BBS hindering them from optimal performance.