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<strong>BACKGROUND</strong>: The V sit-and-reach (VSR) test seems to be an appropriate instrument of self-assessment of hamstring and low-back flexibility for its ease of execution and the need for only a small amount of materials, space, and examination skill requirements. It is assumed that the specificity of self-assessment (in general) can be the cause of other sources of measurement error. <strong>OBJECTIVE</strong>: This study aimed to analyze reliability of VSR when used as an instrument of self-assessment of flexibility in adolescent females. <strong>METHODS</strong>: The sample comprised 43 students (female; age 21.2 ± 0.5 years) from Palacký University in Olomouc (Czech Republic). T-test (p < .05) and Pearson correlation coefficient was used to assess systematic bias and to determine intra-individual reliability of the flexibility test; the standard error of measurement (SEM) and Bland and Altman’s 95% limits of agreement were used to assess absolute reliability of the flexibility test. <strong>RESULTS</strong>: The average intra-individual difference of 1.14 cm (increasing test performance) was found to be statistical significant (t = –5,375; df = 42). It was observed that high intra-individual reliability (r = .98); the absolute reliability (SEM) is equal to 0.139 cm. <strong>CONCLUSIONS</strong>: This study provides evidence supporting the usage of VSR as a relevant instrument of self-assessment of hamstring and low-back flexibility in adolescent females.