The aggregate structures of two di-n-alkyl-phosphate anions (di-n-alkyl: n-butyl(n-dodecyl)(BDoP) and n-hexyl(n-dodecyl)(HDoP)) in water were studied by the use of SANS, cryo-TEM, Raman scattering, C-13 NMR, and selective NOE (H-1{H-1}) techniques. The results of SANS indicated that the different -CH2- lengths of the short chain led to a marked difference in the aggregational behavior of BDoP and HDoP. Cryo-TEM added direct images to support the average aggregation size and shape predicted by the SANS analyses. Raman scattering, C-13 NMR, and selective NOE results provided further evidence that variation of molecular conformations strongly contributed to variation in the shape of the aggregates. In particular, selective NOE was a powerful technique for investigating the dynamic structures of the hydrocarbon chains during growth of the micelles.