The aims of this study were (a) to determine and compare the concurrent hamstring criterion-related validity of the sit-and-reach (SR) and toe-touch (TT) tests in different athletes (tennis players, kayakers, canoeists, and cyclists); (b) to determine the criterion-related validity of the pelvic tilt assessed by the Spinal Mouse system as a measure of hamstring flexibility in athletes; and (c) to evaluate the influence of spinal posture, pelvic tilt, and hamstring muscle flexibility in the SR and TT scores. Twenty-four tennis players, 30 canoeists, 43 kayakers, and 44 cyclists were recruited. Passive straight leg raise (PSLR), SR, and TT tests were randomly performed. Spinal curvatures and pelvic tilt were evaluated with a Spinal Mouse system when the maximal trunk flexion was achieved in the SR and TT tests. Tennis players and cyclists showed moderate correlations between PSLR with respect to SR (β = 0.78 and β = 0.76, respectively) and TT (β = 0.77 and β = 0.74, respectively). Correlations were slightly lower in canoeists (SR, β = 0.64; TT, β = 0.75). Kayakers showed the lowest correlation values (SR, β = 0.53; TT, β = 0.57). Correlation values between PSLR and pelvic tilt angle in both the SR and TT tests were β < 0.70 in all the groups of athletes. Stepwise multiple regression analysis showed a high variance explained from pelvic tilt and lumbar spine in the SR score. In conclusion, the SR and TT tests can be appropriate measures to determine spine flexibility and pelvic tilt range of motion but not to evaluate the hamstring muscle flexibility in tennis players, canoeists, kayakers, and cyclists.