Historically, metaphase chromosomes have been differentiated (or karyotyped), within a species using chemical banding techniques and optical microscopy. This chemical approach alters the structure of the chromosomes. Following successful work on high-resolution imaging of plant chromosomes, the volumes of complete metaphase sets of maize and barley chromosomes have been calculated from three dimensional atomic force microscopy data. These results correlated extremely well with classification by arm lengths and ratios. This study demonstrates the novel application of atomic force microscopy as a high-resolution measuring instrument for volumes of biological systems, a hitherto underused facility of the microscope. This approach will permit chromosomes to be identified and experiments performed on them without recourse to destructive chemical treatment.