Aims. To seek gender differences among the effects of the Graston Technique (GT), Active Release Technique (ART), and Proprioceptive Neuromuscular Facilitation (PNF) stretching on hamstring flexibility, pain, lower extremity function, and physical function in patients with knee osteoarthritis. Materials and methods. A multicenter, single-blind randomized controlled trial was conducted involving 45 patients – aged above 30 years with knee osteoarthritis – randomly allocated to three equal groups: Group-A (GT), Group-B (ART), and Group C (PNF). Each group received 12 physiotherapy sessions over a four-week intervention period (3 visits per week). Data were collected before and after rehabilitation using the Numeric Pain Rating Scale, Active Knee Extension Test, Lower Extremity Function Scale, and Western Ontario and McMaster Universities Arthritis Index (WOMAC). Results. A statistically significant interaction between the effects of gender and Active Release Technique on lower extremity function was present (p < 0.001) with males (42.4 ± 2.2) showed more improvement than females (35.6 ± 6.6). On the contrary, females showed more improvement (p < 0.001) than males when Graston Technique and PNF techniques were applied. Conclusion. There were no significant gender-based differences among GT, ART, and PNF regarding pain, hamstring flexibility and WOMAC score. However, males showed significantly more improvement in lower extremity function than females with ART, and females showed more improvement in lower extremity function as compared to males with GT and PNF.