Gene expression domains are normally not arranged in vertebrate genomes according to their expression patterns. Instead, it is not unusual to find genes expressed in different cell types, or in different developmental stages, sharing a particular region of a chromosome. Therefore, the existence of boundaries, or insulators, as non-coding gene regulatory elements, is instrumental for the adequate organization and function of vertebrate genomes. Through the evolution and natural selection at the molecular level, and according to available DNA sequences surrounding a locus, previously existing or recently mobilized, different elements have been recruited to serve as boundaries, depending on their suitability to properly insulate gene expression domains. In this regard, several gene regulatory elements, including scaffold/matrix-attachment regions, members of families of DNA repetitive elements (such as LINEs or SINEs), target sites for the zinc-finger multipurpose nuclear factor CTCF, enhancers and locus control regions, have been reported to show functional activities as insulators. In this review, we will address how such a variety of apparently different genomic sequences converge in a similar function, namely, to adequately insulate a gene expression domain, thereby allowing the locus to be expressed according to their own gene regulatory elements without interfering itself and being interfered by surrounding loci. The identification and characterization of genomic boundaries is not only interesting as a theoretical exercise for better understanding how vertebrate genomes are organized, but also allows devising new and improved gene transfer strategies to ensure the expression of heterologous DNA constructs in ectopic genomic locations.