Background: Numerous reliable methods are available for evaluating hamstring flexibility, with the sit-and- reach test emerging as the most widely employed approach. Objective: To analyze the intra-rater and inter-rater reliability and the criterion validity of Kinovea software for measuring hamstring extensibility using the sit-and- reach test. Methods: 65 participants, aged between 18 and 25 years, participated in this study. Hamstring extensibility was measured through the sit-and-reach test, concurrently captured by a video camera. Each video underwent downloading onto a computer, followed by analysis using Kinovea software to evaluate criterion validity. Participants received two shots; the 1st shot had been done in the 1st of assessment and the 2nd shot had been done 1 week later to asses intra rater reliability and first assessment had been done by two different examiners to assess inter rater reliability. Results: The correlation coefficients indicated an extremely high correlation between the sit-and-reach test and Kinovea measurements (p < 0.001). Intra-rater reliability was excellent, showing an intraclass correlation coefficient (ICC) of 0.996 for Kinovea measurements. Inter-rater reliability was excellent, showing an ICC of 0.999 for Kinovea measurements. Cohen's Kappa statistic showed almost perfect agreement between raters, with a value of 0.969 for all measurements. Conclusions: The Kinovea software showed excellent intra-rater, inter-rater reliability and strong criterion validity for measuring hamstring extensibility compared to the sit-and-reach test.