The concept of stretching is to increase the range of motion of a joint. Theoretical evidence suggests that the in vivo properties of tendon are different between men and women. There exist gender differences in the viscoelastic properties of a tendon too. This suggests that stretching affects the properties of a muscle and because male and female tendons have different viscoelastic properties, the effect of stretching should also vary accordingly. However, no experimental study has been conducted till date to verify these theoretical constructs. Therefore, the objective of this study was to demonstrate any difference between male and female Rectus Femoris muscle flexibility following application of Proprioceptive Neuromuscular Facilitation hold relax stretching. An experimental comparative study was conducted among 30 students in a physiotherapy college using purposive sampling. Mean values of all the readings of active knee flexion range of motion (AKFROM) at 0, 3rd and 7th day were taken for both the groups. No significant difference was observed between 0 - 3rd and 0 - 7th day but statistically significant results were found between 3rd - 7th day. These findings were more significant among females. It was therefore concluded that the PNF hold-relax stretching has a positive effect on improving Rectus femoris muscle flexibility and this effect is more prominent in females.