Calmodulin (CaM) is a primary calcium (Ca2+)‐signaling protein that specifically recognizes and activates highly diverse target proteins. We explored the molecular basis of target recognition of CaM with peptides representing the CaM‐binding domains from two Ca2+‐CaM‐dependent kinases, CaMKI and CaMKII, by employing experimentally constrained molecular simulations. Detailed binding route analysis revealed that the two CaM target peptides, although similar in length and net charge, follow distinct routes that lead to a higher binding frustration in the CaM–CaMKII complex than in the CaM–CaMKI complex. We discovered that the molecular origin of the binding frustration is caused by intermolecular contacts formed with the C‐domain of CaM that need to be broken before the formation of intermolecular contacts with the N‐domain of CaM. We argue that the binding frustration is important for determining the kinetics of the recognition process of proteins involving large structural fluctuations. Copyright © 2015 John Wiley & Sons, Ltd.