Anion-exchange high performance liquid chromatography (AEX- HPLC) has been widely used to assess the quantity, quality and stability of adenoviral vectors investigated in pre-clinical and clinical gene therapy studies. We have developed an AEX-HPLC method to analyze liquid-stored adenoviral vectors for changes during long- term storage in a variety of formulations at 5° and 25°C. Applying orthogonal methods, we have determined that AEX-HPLC retention time (RT) correlates with changes in physical and functional properties of the virus during storage. Three virus species with distinct physical and biological properties are identifiable by the AEX-HPLC method: (A) the native intact virus population having no RT shift, (B) a population undergoing a gradual rate of RT shift, and (C) a population traversing a phase of rapid RT shift. We have characterized the physical attributes of representative preparations of these three species using reversed-phase (RP) HPLC and quantitative anti-fiber western blots (WB) for capsid-associated viral protein content, picogreen dye binding assay for dye-accessable viral dsDNA content, and electron microscopy. In addition, we assessed the biological activity of the viral species by a virus-cell attachment assay, transgene expression assay and plaque assay. Our results show species A and B to be virions with complete vertices, termed “intact-capsid virions (ICV),” and fully biologically functional. Species B, although intact and fully functional, displays a modified capsid surface charge of increasing acidity, suggestive of surface protein deamidation. Specie C, though still containing the viral DNA core, is devoid of fiber and penton base complex (penton), and has consequently lost its cell-attachment function, thus is non-infectious. Accordingly, specie C has been termed “penton-vacant virion (PVV).” In conclusion, the retention time attribute of AEX-HPLC is sufficient to differentiate native ICV, deamidated ICV, and PVV populations as they occur along the instability pathway of liquid-stored adenoviral vectors.