This paper reports the findings of an investigation into the molecular structures and properties of three asphaltene samples, namely, an asphaltene sample extracted from Buton Oil Sand (Indonesia), and two asphaltene samples extracted from vacuum residues from Liaohe Refinery (China) and Vene Refinery (Venezuela), respectively. The average molecular structural parameters, including the average polycyclic aromatic hydrocarbon (PAH) size, average side chain length, and average molecular weight (AMW), of the three asphaltene samples were estimated using data from nuclear magnetic resonance (NMR) in combination with distortionless enhancement by polarization transfer (DEPT), and then compared against each other. The molecular weight distributions (MWDs) of the three asphaltene samples were measured using a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The results indicated that the island molecular architecture predominated in all three asphaltenes and the average polycyclic aromatic hydrocarbon size was found to be six rings. The average molecular weight of the Buton asphaltene sample was found to be ca. 800 Da while those of the two petroleum asphaltene samples were approximately 600 Da. In comparison, the Buton asphaltene sample contained a much higher level of oxygen and sulfur, but a lower aromaticity than those of the two petroleum asphaltene samples. The use of liquid NMR in combination with DEPT was shown to provide an effective method for characterization and estimation of the molecular structures of asphaltenes, supported by MALDI-TOF mass spectra.