Article chromosome organization in higher eukaryotes controls gene expression, dna replication, and dna repair. genome mapping has revealed the functional units of chromatin at the submegabase scale as self-interacting regions called topologically associating domains (tads) and showed they correspond to replication domains (rds). a quantitative structural and dynamic description of rd behavior in the nucleus is, however, missing because visualization of dynamic subdiffraction-sized rds remains challenging. using fluorescence labeling of rds combined with correlative live and super-resolution microscopy in situ, we determined biophysical parameters to characterize the internal organization, spacing, and mechanical coupling of rds. we found that rds are typically 150 nm in size and contain four co-replicating regions spaced 60 nm apart. spatially neighboring rds are spaced 300 nm apart and connected by highly flexible linker regions that couple their motion only <550 nm. our pipeline allows a robust quantitative characterization of chromosome structure in situ and provides important biophysical parameters to understand general principles of chromatin organization