Abstract: The molecular structure of second generation hydroxyl terminated aliphatic hyperbranched polyester (HB-20) was characterized by nuclear magnetic resonance (NMR) analyses, including one-dimensional (1D) NMR technique (¹H-NMR, ¹³C-NMR and distortionless enhancement by polarization transfer (DEPT) NMR spectra) and two-dimensional (2D) NMR technique (heteronuclear single quantum coherence (HSQC) NMR and heteronuclear multipe bond coherence (HMBC) NMR spectra). Firstly, the peaks of the ¹H-NMR and ¹³C-NMR spectra were initially assigned, and the four types of carbon atoms (CH₃, CH₂, CH, and quaternary carbon atom C^*) in the HB-20 were distinguished by DEPT NMR spectra. Secondly, the detailed molecular structure information was further obtained by 2D NMR analysis. From the HSQC spectrum, the ¹³C-¹H chemical shift correlation spectroscopy spectrum contained all the directly linked ¹³C-¹H coupling relationships, and the positions of the related ¹H and ¹³C in the molecular structure were determined; from the HMBC spectrum, the ¹³C and ¹H coupling information separated by 2-3 chemical bonds was obtained, and the C-C correlation was confirmed to determine the molecular skeleton information of HB-20. According to the NMR analysis, the three relative peak areas of the confirmed T-CH₃, L-CH₃, and D-CH₃ structures in the ¹H-NMR spectrum were determined to be 2.55, 3.97 and 1.00 respectively, and the average degree of branching (DB) of HB-20 was calculated as 0.47. Moreover, the average molecular weight of HB-20 was calculated as 1 876 by NMR analysis, which was closed to that obtained from the hydroxyl value titration, about 1 878. In summary, the molecular structure of HB-20 was clearly characterized.