Two kinds of amphiphilic polypeptoids having different types of hydrophilic polypeptoids, poly(sarcosine)-b-(L-Leu-Aib)(6) (ML12) and poly(N-ethyl glycine)-b-(L-Leu-Aib)(6) (EL12), were self-assembled via two paths to phase separated nanotubes. One path was via sticking ML12 nanotubes with EL12 nanotubes and the other was a preparation from a mixture of ML12 and EL12 in solution. In either case, nanotubes showed temperature-induced phase separation along the long axis, which was observed by two methods of labeling one phase with gold nanoparticles and fluorescence resonance energy transfer between the components. The phase separation was ascribed to aggregation of poly(N-ethyl glycine) blocks over the cloud point temperature. The addition of 5% trifluoroethanol was needed for the phase separation because the tight association of the helices in the hydrophobic region should be loosened to allow lateral diffusion of the components to be separated. The phase separation in molecular assemblies in water based on the hydrophilic-region-driven-type mechanism therefore requires sophisticated balances of association forces exerting among the hydrophilic and hydrophobic regions of the amphiphilic polypeptoids.