Fission gas release from high-burnup mixed-oxide (MOX) fuel pellet with Pu fissile contents of 4.1% during reactivity-initiated accident (RIA) was estimated based on radial Xe concentration profiles in fuel pellet using scanning electron microscopy/electron probe microanalysis (SEM/EPMA) before and after pulse irradiation tests called BZ-3 and BZ-4. The tests were conducted at the Nuclear Safety Research Reactor (NSRR) in Japan Atomic Energy Agency (JAEA). The depression of Xe concentration in the Pu-spot and the crack formation in the peripheral region of fuel pellet were observed after BZ-3 and BZ-4. This kind of cracks was considered to promote the release of the fission gas retained in the Pu-spot. Based on the EPMA results and the effect of the crack formation, the amounts of the release of the gas retained in Pu-spot was estimated to be 0.13 and 0.04 in BZ-3 and BZ-4 tests, respectively. Moreover, taking account of the release of the gas accumulated in the grain boundary in Pu-spot-excluded region and the release of the gas dissolved in the matrix, the total amount of fission gas release was evaluated as 0.33–0.43 and 0.15–0.27 in BZ-3 and BZ-4 tests, respectively. Comparing the results of the fission gas release with the results of rod-puncture test, it was suggested that fission gas release from not only the Pu-spot but also the Pu-spot-excluded region.