Unilateral osteoarthritis (OA) affects single knees and presents a unique scenario where individuals experience varying degrees of symptoms between their affected and unaffected knees. This study aims to investigate differences in muscle tightness between symptomatic and asymptomatic knees in individuals with unilateral knee OA while exploring the interplay among pain, functionality, and muscle tightness. In this cross-sectional study, thirty knee OA patients underwent assessments for hamstring (Active Knee Extension, Straight Leg Raise), iliotibial band (Ober Test), and quadriceps tightness (Modified Thomas Test). Pain intensity was measured using the Visual Analog Scale (VAS), and functional limitations were evaluated via the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC). A negative correlation was observed between participants' pain and AKE (p= 0.004, r=-0.515), ASLR (p= 0.27, r=-0.403), Ober (p= 0.010, r=-0.461) values. However, no significant correlation was found with the Modified Thomas value (p= 0.204, r=-0.239). There was also a negative correlation between participants' WOMAC scores and AKE (p= 0.019, r=-0.427), OBER (p= 0.004, r=-0.510), and Modified Thomas (p= 0.022, r=-0.416) values, while ASLR (p= 0.286, r=-0.202) values showed no significant correlation. Comparisons between AKE, Ober, and Modified Thomas values showed higher values in asymptomatic extremities (AKE: p= 0.025, Ober: p= 0.021, Modified Thomas: p= 0.030). This study emphasizes the significance of muscle tightness in the symptomatic extremities of individuals with unilateral knee OA. The results indicate that increased muscle tightness makes pain worse and limits movement. It's crucial for healthcare providers treating OA to focus on improving muscle flexibility, reducing pain, and enhancing overall function.