Fluorescence measurements from metaphase chromosomes of the Chinese hamster, stained with propidium iodide excited at high illumination irradiance, completely resolve each chromosome type. The measurements are performed in a specially designed flow cytometer that achieves high irradiance (4 MW/cm2) by using high power laser output (2 W at 488 nm) focused to small spot size (1% irradiance variation over 2 microns). The coefficient of variation of each chromosome peak is near 1.5%. Saturation of the fluorescence transition and photobleaching, two consequences of high irradiance, are shown to occur. Even with a nonlinear dependence of fluorescence upon illumination irradiance, fluorescence retains a proportional response to chromosome type; each chromosome peak maintains a consistent ratio to the others at every irradiance. No perturbation of fluorescence by the optical or geometrical properties of the chromosomes is evident. The advantages of high irradiance illumination are an increase in fluorescence sufficient to reduce the statistical error in photoelectron number to a low level and reduced influence of laser power fluctuations and variable chromosome flow trajectories on the precision. These benefits improve the resolution of chromosome analysis by flow cytometry, particularly the resolution of smaller chromosomes.