The eukaryotic genome is packaged as chromatin within the three-dimensional nuclear space. Decades of cytological studies have revealed that chromosomes and genes are non-randomly localized within the nucleus and such organizations have important roles on genome function. However, several fundamental questions remain to be resolved. For example, what is required for the preferential localization of a gene to a nuclear landmark? What is the mechanism underlying gene repositioning in the nucleus? How does subnuclear gene positioning regulate gene transcription? Recent studies have revealed that several factors such as DNA sequence composition, specific regulatory sequences, epigenetic modifications, chromatin remodelers, post-transcriptional regulators and nuclear architectural proteins can influence chromatin dynamics and gene positioning in a gene-specific manner among organisms from yeast to human. In this review, we discuss some recent findings as well as experimental tools to investigate subnuclear gene positioning and to explore its implications in genome functions.