Localized magnetic heating treatments (hyperthermia, thermal ablation) using superparamagnetic iron oxide nanoparticles (MNPs) continue to be an active area of cancer research. For generating the appropriate heat to sufficiently target cell destruction, adequate MNP concentrations need to be accumulated into tumors. Furthermore, the knowledge of MNP bio-distribution after application and additionally after heating is significant, firstly because of the possibility of repeated heating treatments if MNPs remain at the target region and secondly to study potential adverse effects dealing with MNP dilution from the target region over time. In this context, little is known about the behavior of MNPs after intra-tumoral application and magnetic heating. Therefore, the present in vivo study on the bio-distribution of intra-tumorally injected MNPs in mice focused on MNP long term monitoring of pre and post therapy over seven days using multi-channel magnetorelaxometry (MRX). Subsequently, single-channel MRX was adopted to study the bio-distribution of MNPs in internal organs and tumors of sacrificed animals. We found no distinct change of total MNP amounts in vivo during long term monitoring. Most of the MNP amounts remained in the tumors; only a few MNPs were detected in liver and spleen and less than 1% of totally injected MNPs were excreted. Apparently, the application of magnetic heating and the induction of apoptosis did not affect MNP accumulation. Our results indicate that MNP mainly remained within the injection side after magnetic heating over a seven-days-observation and therefore not affecting healthy tissue. As a consequence, localized magnetic heating therapy of tumors might be applied periodically for a better therapeutic outcome.