Maternal and paternal homologous chromosomes recognize each other and pair during meiosis to ensure accurate chromosome segregation. However, the mechanisms that drive chromosome pairing and their regulation remain poorly understood. To investigate this, we develop a fluorescence reporter-operator system based on the lac operator-lac repressor system, enabling real-time observation of specific genomic loci in live mouse seminiferous tubule tissues. Using 3D time-lapse imaging, we visualize and quantify telomere-led rapid chromosome movements and homologous chromosome pairing. Our results reveal that homologous chromosomes achieve pairing in discrete, well-defined steps. Initially, at the onset of meiosis, chromosomes come together from widely separated positions at an average distance of 4.8 µm, followed by an intermediate stage of pairing at an average distance of 1.7 µm. Finally, close pairing is achieved at an average distance of 0.7 µm, which correlates with the establishment of the synaptonemal complex along the homologs. Further analysis of inter-spot distance dynamics reveals a relatively rapid transition from an initial, widely separated distance to an intermediate stage of chromosome pairing. Complementary studies of recombination mutants using 3D DNA fluorescence in situ hybridization (FISH) on whole-mounted tubules and immunofluorescence on chromosome spreads reveal that double-strand breaks and early recombination events trigger the transition from a widely separated to an intermediate inter-homolog distance. Subsequently, close pairing requires crossover maturation intermediates and SYCP1/synaptonemal complex. Together, our findings reveal a recurring mechanism of meiotic chromosome pairing and how it is regulated by critical meiotic processes in higher eukaryotes.