In cultured cells, endogenous or ectopically expressed p62 forms cytoplasmic inclusion bodies (p62 bodies) (protein localization, particle size and count by microscopy). p62 bodies contain polyubiquitin chains and K63 polyubiquitin chains are preferentially recruited into p62 bodies (protein co-localization). In p62-GFP-expressing autophagy-defective Atg12−/− cells (genetic transformation, knock-out, overexpression, fusion protein), p62 bodies were spherical and could undergo fusion (morphology). Furthermore, FRAP experiments revealed that the fluorescent signal recovered after bleaching of p62 bodies. No phase separation occurs in a solution containing even as much as 120 µM recombinant mCherry-fused p62 in vitro (particle size and count by microscopy), only when adding a solution of K63 polyubiquitin chains. In the control reaction, which did not contain adenosine triphosphate (ATP) and therefore did not form K63 polyubiquitin chains, p62 phase separation was not induced. Thus, K63 polyubiquitin chains, but not monoubiquitin can induce p62 phase separation. When adding cytosol from p62−/− cells to recombinant mCherry-fused p62 in vitro LLPS occured (particle size and count by microscopy). However treatment of the p62−/− cytosol with UPS5, a deubiquitinating enzyme that removes polyubiquitin chains abolished in vitro LLPS. Polyubiquitinated proteins are segregated into p62 droplets (co-localization). Although both Ubx8 and Ubx6 could cause p62 phase separation, Ubx8 could induce p62 phase separation at a much lower concentration; moreover, when the concentrations of both p62 and Ub were fixed, Ubx8 induced much stronger p62 phase separation than Ubx6, thus the valence of the polyubiquitin chain is important for p62 LLPS. LC3 co-localized with p62 bodies in Atg12−/− cells (it could be recruited to the p62 bodies) in vivo and also showed similar behaviour in vitro. In vivo polyubiquitin chain-induced p62 phase separation (particle size and count by microscopy) was markedly impaired by mCherry-p62-M404V and mCherry-p62-ΔPB1 mutations. Phosphorylation of S403 promotes polyubiquitin chain-induced phase separation, p62 body formation, and autophagic degradation (particle size and count by microscopy). The M404T and G411S Paget’s disease of bone mutations both markedly impaired polyubiquitin chain-induced p62 phase separation (particle size and count by microscopy).