Lysosomal targeting of ubiquitylated endocytic cargo is mediated in part by the endosomal sorting complex required for transport (ESCRT) complexes, a system conserved between animals and fungi (Opisthokonta). Extensive comparative genomic analysis demonstrates that ESCRT factors are well conserved across the eukaryotic lineage and complexes I, II, III and III-associated are almost completely retained, indicating an early evolutionary origin. The conspicuous exception is ESCRT 0, which functions in recognition of ubiquitylated cargo, and is restricted to the Opisthokonta, suggesting that a distinct mechanism likely operates in the vast majority of eukaryotic organisms. Additional analysis suggests that ESCRT III and ESCRT III-associated components evolved through a concerted model. Functional conservation of the ESCRT system is confirmed by direct study in trypanosomes. Despite extreme sequence divergence, epitope-tagged ESCRT factors TbVps23 and TbVps28 localize to the endosomal pathway, placing the trypanosome multivesicular body (MVB) in juxtaposition to the early endosome and lysosome. Knockdown of TbVps23 partially prevents degradation of an ubiquitylated endocytosed transmembrane domain protein. Therefore, despite the absence of an ESCRT 0 complex, the trypanosome ESCRT/MVB system functions similarly to that of opisthokonts. Thus the ESCRT system is an ancient and well-conserved feature of eukaryotic cells but with key differences between diverse lineages.