A stock of tobacco with 23 pairs of tobacco chromosomes and one substituted pair from Nicotiana glutinosa was available. An amphiploid of this tobacco with N. tomentosiformis was synthesized in order to test whether preferential pairing is determined by the homologies of the chromosomes or whether it is under genic control. Characteristically, segregates for duplex loci in N. tabacum × N. tomentosiformis amphiploids give a gametic output of about 3:1, but for a factor on the substituted chromosome that output was found to be 59:1. The result suggests that preferential pairing in this material is not genically determined.