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A series of new waterborne polyurethanes (WPUs) was successfully prepared by prepolymer process from renewable source, hydroxytelechelic natural rubber (HTNR), with different amounts of DMPA (1.6-8.4 wt %), different molecular weights (1000-4000 g mol−1), and different levels of epoxide (0%-20%) of HTNR. It was found that the urethane conversions of prepolymer were over 80% as calculated by FTIR technique. The resulting HTNR2000-based WPUs exhibit a uniform particle size, which decreases from 420- to 83-nm diameters with an increase in the amount of DMPA from 2.9 to 6.6 wt %. The particle size also decreases with an increase of soft segment or with an increase of epoxide content. They are well stable more over 6 months and without a significant difference in particle size compared with starting of them. Chemical structure of WPU films was confirmed by FTIR, 1H-NMR, and 13C-NMR. Molecular weight and polydispersity were determined by SEC. In addition, thermal and water uptake properties were investigated. The experimental results reveal that the DMPA content, molecular weight of HTNR, and epoxide content play an important key role in water uptake and thermal properties. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012 [ABSTRACT FROM AUTHOR]