To prepare water-borne polyurethane dispersions (PUD) from novel renewable-based, asymmetric bifunctional building blocks, it is important to understand the reactivity and regio-selectivity differences between the various functional groups and reagents, respectively. This paper firstly describes the mutual reactivity and regio-selectivity of biomass-derived, asymmetric 1,4:3,6-dianhydro-D-glucitol (isosorbide, IS) and ethyl ester L-lysine diisocyanate (EELDI) in polyurethane (PU) syntheses. The regio-selectivities of the endo- and the exo-OH functional groups of IS and the primary e-NCO and the secondary a-NCO of EELDI were found to have only minor consequences for the formation of NCO-terminated PU prepolymers. In addition, a model study of IS, dimethylolpropionic acid (DMPA), EELDI and a dimer fatty acid-based diisocyanate (DDI) in their respective PU reactions revealed the reaction rate differences between these four compounds. Surprisingly, a comparatively low reaction rate of DMPA was observed. For the PU prepolymers synthesized from the four mentioned components, an enrichment in DMPA near the polymer chain ends may thus be expected. Finally, PU dispersions prepared from these four-component prepolymers showed a good storage stability. The relatively small particle size at a low DMPA content and a high EELDI and IS content is predominantly regarded as the result of the hydrophilicity of EELDI, IS and possibly the enhanced DMPA concentration near the chain ends of the PU prepolymer.