3D genomics mainly focuses on the 3D position of single genes at cell level, while spatial genomics focuses more on tissue level. In this exciting new era of 3D/spatial genomics,, half-century old FISH and its derivative methods, including Tn5-FISH, play important role. In this review, we introduce the Tn5-FISH we developed recently, and present six different applications published by our collaborators and us, based on (Tn5-)FISH, which can be either general BAC clone-based FISH or Tn5-FISH. In these interesting cases, (Tn5-)FISH demonstrated its vigorous ability of targeting sub-chromosomal structure across different diseases and cell lines (leukemia, mESCs(mouse Embryonic Stem Cells), and differentiation cell lines). Serving as an effective tool to image genomic structures at kilobase level, Tn5-FISH holds great potential to detect chromosomal structures in a high-throughput manner, thus bring the dawn for the new discoveries in the great era of 3D/spatial genomics.