ABSTRACTComparing a parasitic lineage to its free-living relatives is a powerful way to understand how the evolutionary transition to parasitism occurred.Giardia intestinalis(Fornicata) is a leading cause of gastrointestinal disease world-wide and is famous for its unusual complement of cellular compartments, such as having peripheral vacuoles instead of typical endosomal compartments. Endocytosis plays an important role inGiardia’s pathogenesis. Endosomal sorting complexes required for transport (ESCRT) are membrane-deforming proteins associated with the late endosome/multivesicular body (MVB). MVBs are ill-defined inG. intestinalisand roles for identified ESCRT-related proteins are not fully understood in the context of its unique endocytic system. Furthermore, components thought to be required for full ESCRT functionality have not yet been documented in this species.We used genomic and transcriptomic data from several Fornicata species to clarify the evolutionary genome streamlining observed inGiardia, as well as to detect any divergent orthologs of the Fornicata ESCRT subunits. We observed differences in the ESCRT machinery complement betweenGiardiastrains. Microscopy-based investigations of key components of ESCRT machinery such asGiVPS36andGiVPS25 link them to peripheral vacuoles, highlighting these organelles as simplified MVB equivalents. Unexpectedly, we show ESCRT components associated with the Endoplasmic Reticulum, and for the first time, mitosomes. Finally, we identified the rare ESCRT component CHMP7 in several fornicate representatives, includingGiardia, and show that contrary to current understanding, CHMP7 evolved from a gene fusion of VPS25 and SNF7 domains, prior to the last eukaryotic common ancestor, over 1.5 billion years ago. Our findings show that ESCRT machinery inG. intestinalisis far more varied and complete than previously thought, and associating to multiple cellular locations and presenting changes in ESCRT complement which pre-date adoption of a parasitic lifestyle.