Accurate estimates of hamstrings lengths are useful, for example, to facilitate planning for surgical lengthening of the hamstrings in patients with cerebral palsy. In this study, three models used to estimate hamstrings length (M1: Delp, M2: Klein Horsman, M3: Hawkins and Hull) were evaluated. This was done by determining whether the estimated peak semitendinosus, semimembranosus and biceps femoris long head lengths, as measured in eight healthy subjects, were constant over a range of hip and knee angles. The estimated peak hamstrings length depended on the model that was used, even with length normalized to length in anatomical position. M3 estimated shorter peak lengths than M1 and M2, showing that more advanced models (M1 and M2) are more similar. Peak hamstrings length showed a systematic dependence on hip angle for biceps femoris in M2 and for semitendinosus in M3, indicating that either the length was not correctly estimated, or that the specific muscle did not limit the movement. Considerable differences were found between subjects. Large inter-individual differences indicate that modeling results for individual subjects should be interpreted with caution. Testing the accuracy of modeling techniques using in vivo data, as performed in this study, can provide important insights into the value and limitations of musculoskeletal models.