Three-dimensional (3D) genome topology establishes blueprints of gene expression. We recently reported the identification, from ensemble Hi-C data, of long-range interactions between multiple non-contiguous TADs, forming repressive cliques of 3-11 TADs (1). We now combine Hi-C, 3D genome modeling and microscopy analyses to provide evidence of multi-TAD assemblies in single cells. First, we determined whether TAD cliques can be detected in single-cell Hi-C datasets (2). Because chromosomal interactions mapped in such datasets are too sparse to directly infer TAD cliques, we computed the contact frequency of TADs found in cliques in corresponding ensemble Hi-C data. We find, for all single cells queried, a significant enrichment of inter-TAD contacts in cliques relative to TADs in a random configuration on the same chromosome. Second, in each single-cell dataset, TAD contact density within cliques indicates that although these cliques are not fully connected in single cells, they display > 50% of TAD-TAD contacts mapped in ensemble data. Thus TADs in cliques display higher connectivity in single cells than what is expected from random TAD encounters. Third, 3D genome structure modeling (3) reveals that co-localization of TADs in cliques, defined by colocalization thresholds, is greater than that of TADs in a random configuration. This implies that long-distance inter-TAD interactions seen in Hi-C data reflect spatial associations between TADs also in single cells. Fourth, this inference is quantitatively supported by fluorescence in situ hybridization analyses of TADs in cliques and outside cliques simultaneously in individual cells. FISH data also reveal heterogeneity in the spatial distribution of TADs in cliques between cells, as well as inter-allelic variation. Our data suggest that non-random assemblies of TADs into cliques shape higher-order 3D genome architecture in single cells. Molecular processes underlying TAD clique dynamics are under investigation.