The aggregation behavior of the bolaamphiphilic hydrogelators dotriacontane-1,32-diyl-bis[2-(trimethylammonio)ethylphosphate] (PC-C32-PC) and the pH-sensitive dotriacontane-1,32-diyl-bis[2-(dimethylammonio)ethylphosphate] (Me2PE-C32-Me2PE) was characterized before using different methods. Dependent on the temperature, pH, and concentration, these bolaamphiphiles self-asssemble into long nanofibers or other aggregates, such as short rods or micelles. In order to obtain information about the motional dynamics and microscopic order inside of these aggregates, we carried out a systematic ESR spin probe study. The spectra obtained with the spin probes 5-, 12- and 16-doxyl stearic acid (n-DSA) were found to be highly sensitive to changes in the bolaamphiphilic arrangement. Rotational correlation times and order parameters were obtained from full ESR line shape simulations with the programs EasySpin and EPRSIM. The maximum hyperfine splitting, 2A(max), was used to determine the transition temperatures, which are in agreement with the DSC data. By comparison of 5-DSA and 12-DSA, which are residing at different positions in the alkyl chain region of the aggregates, it was found that trans-gauche isomerization is predominantly occurring in the outer region of the aggregates. For Me2PE-C32-Me2PE at pH 10, our ESR data indicate that micelles are short rods rather than being spherical in shape. Therefore, increasing the concentration from 1 to 10 mg/mL leads only to a one-dimensional growth of these micelles.