The current study evaluates the cytogenetic effects of chromium (III) oxide nanoparticles on the root cells of Allium cepa. The root tip cells of A. cepa were treated with the aqueous dispersions of Cr2O3 nanoparticles (NPs) at five different concentrations (0.01, 0.1, 1, 10, and 100 μg/mL) for 4 hr. The colloidal stability of the nanoparticle suspensions during the exposure period were ascertained by particle size analyses. After 4 hr exposure to Cr2O3 NPs, a significant decrease in mitotic index (MI) from 35.56% (Control) to 35.26% (0.01 μg/mL), 34.64% (0.1 μg/mL), 32.73% (1 μg/mL), 29.6% (10 μg/mL) and 20.92% (100 μg/mL) was noted. The optical, fluorescence and confocal laser scanning microscopic analyses demonstrated specific chromosomal aberrations such as—chromosome stickiness, chromosome breaks, laggard chromosome, clumped chromosome, multipolar phases, nuclear notch, and nuclear bud at different exposure concentrations. The concentration-dependent internalization/bio-uptake of Cr2O3 NPs may have contributed to the enhanced production of anti oxidant enzyme, superoxide dismutase to counteract the oxidative stress, which in turn resulted in observed chromosomal aberrations and cytogenetic effects. These results suggest that A. cepa root tip assay can be successfully applied for evaluating environmental risk of Cr2O3 NPs over a wide range of concentrations.