Heat shock protein 40s (Hsp40s), also known as J-proteins, are conserved in prokaryotes and eukaryotes. The Zuotin/Zuotin-related factor (ZUO1/ZRF) family belongs to a novel Hsp40 clade exclusively found in eukaryotes. Zuotin/Zuotin-related factor proteins are characterized by a large N terminal ZUO1 domain originally identified in the yeast ZUO1 protein. The ZUO1 domain is characterized by a highly conserved J-domain, together with an atypical UBD domain first identified in the human ZRF1 protein. Furthermore, ZUO1/ZRF protein families in animals and plants harbor a pair of C terminal SANT domains, suggesting the divergence of their functions with those in fungi. Zuotin/Zuotin-related factor proteins retain the ancestral function as an Hsp70co-chaperone implicated in protein folding and renaturation after stress; these proteins also perform diverse neofunctions in the cytoplasm and transcriptional and/or epigenetic regulatory functions in the nucleus. Therefore, these proteins are involved in translational fidelity control, ribosomal biogenesis, asymmetric cell division, cell cycle, apoptosis, differentiation, and tumorigenesis. The results of sequence and domain organization analysis of proteins from diverse organisms provided valuable insights into the evolutionary conservation and diversity of ZUO1/ZRF protein family. Further, phylogenetic analysis provides a platform for future functional investigation on the ZUO1/ZRF protein family, particularly in higher plants.