The alpha- and beta-globin gene clusters are subject to several levels of regulation leading to differentiation and developmental stage-specific expression. Not only the interaction of cis-acting elements and cell-specific trans-acting factors but also the chromosomal environment, nuclear compartmentalization of genome regions, and transcription factors play important roles in globin gene regulation research. FISH, with a refined technique and an incorporation of other molecular techniques, has become a powerful technique in gene regulation research, particular for the analysis of replication timing and primary transcripts as well as the research of the special organization of large-scale chromatin domains and nuclear compartmentalization at cellular and subcellular levels. This review will focus on the major impact of FISH on globin gene regulation research at different regulation levels, such as replicating timing, gene transcription, nuclear relocation, and chromosomal position effect.