Eleven generations of bidirectional selection for lateralization produced 2 lines of mice that differ markedly in degree of asymmetry for hand preference. The foundation population was derived from 6 distantly related inbred strains and 2 stocks of wild mice, M. castaneus. HI line matings were made using mice that exhibited consistent right or left paw use in a food reaching task and LO line matings were made using mice with little overall paw preference. All matings were made without regard to the expressed directions of asymmetry. Line differences emerged at the third generation and increased thereafter. Selection was relaxed at generation 12 and the lines were maintained by random within-line mating. At generation 28 selective breeding was reimpressed for 3 generations. Results indicated that between-line divergence in degree of lateralization had remained high during 17 generations of relaxed selection. Mice of the HI line are more strongly lateralized than mice of the unselected HET population. Mice of the LO line are more weakly lateralized than controls. The selected lines may provide a useful mammalian genetic resource for studying the neurobiology of cerebral lateralization.