Microwave-assisted thermal sterilization (MATS) is an advanced thermal process that utilizes microwave (MW) energy for in-package food sterilization. Benefits include much shorter processing times than conventional retort sterilization. This research explores how MATS affects the performance of high-barrier multilayer polymeric films compared with conventional retort sterilization. The gas barrier, morphological, and free volume packaging properties of these films may influence the shelf-life of shelf-stable foods. In this study, we applied X-ray diffraction (XRD) and positron annihilation lifetime spectroscopy in order to investigate film morphology and free volume characteristics, respectively. Results show that the conventional retort process affected gas barrier properties more than MATS processing did which could be explained by the morphological and free volume changes in the polymeric films. XRD revealed improved crystalline morphology of MW-treated films in terms of overall crystallinity as compared with retort sterilization. On the other hand, higher free volume increase in MW-treated films could be explained by the different heating mechanisms involved in MATS and retort sterilization. Overall, the oxygen transmission rate for both films remained below 2 cc/m2-day after MATS and retort sterilization required for packaging applications for shelf-stable foods. This work provides the basis for understanding the gas-barrier changes of multilayer polymeric films after MATS application using Materials Science techniques. © 2014 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2014, 131, 40376.