Several approaches for predicting secondary structures from sequences have been developed and reached a fair accuracy. One of the most rigorous tests for these prediction methods is their ability to correctly predict identical fragments of protein sequences adopting different secondary structures in unrelated proteins. In our previous work, we obtained 30 identical octapeptide sequence fragments adopting different backbone conformations. It is of interest to find whether the presence of structurally ambivalent fragments in proteins will affect the accuracy of secondary structure prediction methods or not. Hence, in this work, we have made a systematic comparative analysis on secondary structure prediction results of 30 identical octapeptide pairs and 52 identical heptapeptide pairs adopting different conformations with the aid of segment overlap measure. The results reveal the better performance of profile-based methods such as PSIpred and JPred and misprediction by classical rule-based methods such as Garnier Osguthorpe Robson Method and Double Prediction Method. The results discussed here insist that modern secondary structure prediction methods are able to better discriminate conformationally ambivalent peptide fragments. © 2012 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 100: 148–153, 2013.