Among the morsitans-group of tsetse there are several pairs of taxa in which there is a marked hybridization asymmetry (HA), i.e., one cross produces significantly more offspring than does the reciprocal cross. To investigate the relative contribution of maternally inherited factors (MIF) and chromosomal factors to HA, three hybrid lines were established in which flies have MIF from one taxon and chromosomes from another. HA was then compared among crosses of the parental taxa and crosses of each parental taxon with the appropriate hybrid line. The results indicate that HA in reciprocal crosses of Glossina morsitans morsitans Westwood and Glossina swynnertoni Austin and in reciprocal crosses of G. m. morsitans and Glossina morsitans centralis Machado are caused by chromosomal factors, not MIF. Reciprocal crosses of G. m. centralis and G. swynnertoni do not display HA, and none developed as a result of a novel combination of MIF and tsetse chromosomes.