There is an increasing interest in understanding how three-dimensional (3D) organization of the genome is regulated.Different strategies have been employed to identify genome-wide chromatin interactions.However,due to current limitations in resolving genomic contacts,visualization and validation of these genomic loci with sub-kilobase resolution remain unsolved to date.Here,we describe Tn5 transposase-based Fluorescence in situ hybridization (Tn5-FISH),a PCR-based,cost-effective imaging method,which can co-localize the genomic loci with sub-kilobase resolution,dissect genome architecture,and verify chromatin interactions detected by chromatin configuration capture (3C)-derived methods.To validate this method,short-range interactions in keratin-encoding gene (KRT) locus in topologically associated domain(TAD) were imaged by triple-color Tn5-FISH,indicating that Tn5-FISH is very useful to verify short-range chromatin interactions inside the contact domain and TAD.Therefore,TnS-FISH can be a powerful molecular tool for the clinical detection of cytogenetic changes in numerous genetic diseases such as cancers.