A measurement of jet substructure observables is presented using tt events in the lepton+jets channel from proton-proton collisions at s=13 TeV recorded by the CMS experiment at the LHC, corresponding to an integrated luminosity of 35.9 fb-1. Multiple jet substructure observables are measured for jets identified as bottom, light-quark, and gluon jets, as well as for inclusive jets (no flavor information). The results are unfolded to the particle level and compared to next-to-leading-order predictions from powheg interfaced with the parton shower generators pythia 8 and herwig 7, as well as from sherpa 2 and Dire 2. A value of the strong coupling at the Z boson mass, αS(mZ)=0.115-0.013+0.015, is extracted from the substructure data at leading-order plus leading-log accuracy.