Although there is some evidence that handedness is associated with structural and functional differences in the motor cortex, findings remain inconclusive. Here, we evaluated whether handedness influences the location, size and overlap of the cortical representations of upper limb muscles across hemispheres in right- versus left-handed individuals. Using transcranial magnetic stimulation, the cortical representations of abductor pollicis brevis, flexor carpi radialis and biceps brachii muscles were mapped bilaterally with a 6 by 5 grid space. Results indicate that right-handers had more lateral and posterior representations in the non-dominant hemisphere as well as greater overall cortical territory compared to left-handers. Right- and left-handers did not differ in the extent of overlap between muscle representations. Our findings suggest that human motor cortical organization of upper limb muscles is indeed influenced by handedness, specifically with regard to the location of non-dominant cortical muscle representations and the size of cortical territory dedicated to upper limb muscle representations.