Primary hyperoxaluria type I (PH1) is a genetic disease caused by a deficiency in the peroxisomal alanine:glyoxylate aminotransferase (AGT) activity. Mutations in AGT mostly cause protein mistargeting and enhanced aggregation, although the molecular and structural basis of these mechanisms are unknown. In this work, we use hydrogen-deuterium exchange monitored by mass spectrometry (HDX-MS) to provide novel insight into these pathogenic mechanisms. We characterize the wild-type (WT) protein, the LM variant (containing the mutations P11L and I340M, a haplotype more frequent in PH1 patients) and the LM G170R (the most common genotype in PH1, introducing the G170R mutation on the LM background). Our study provides the first experimental analysis of the local stability and dynamics of AGT, showing that stability is heterogeneous in the native state and providing a blueprint for frustrated regions with potentially functional relevance. The LM and LM G170R variants destabilize locally the structure. Enzymatic transamination of the PLP bound to AGT hardly affects stability. Our study thus supports that AGT misfolding is not caused by dramatic effects on the stability and dynamics of the holo-protein.