In this paper, the chain packing and phase transition of comb-like polymer has been deeply analyzed with N-hexacosylated polyethyleneimine (PEI26C) as a template, fabricated through the reaction between n-hexacosyl bromide and PEI in a homogenous solution. The effect of long alkyl groups on the chain packing and phase transition of PEI26C was systematically investigated by synchrotron X-ray scattering and variable-temperature FTIR spectroscopy. PEI26C comb-like polymer exhibited an interesting structure-evolution process, and phase transformation from orthorhombic (beta(O)), monoclinic (beta(M)), to hexagonal (alpha(H)) phase, and finally to amorphous state was demonstrated, indicating that large alkyl domains induced the complicated structures. Size-depended phase transition behavior provides an insight into the formation of metastable structure during the early stage of polymer crystallization. (C) 2013 Elsevier Ltd. All rights reserved.