Non-viable hybrid seedlings were produced from a cross between Nicotiana tabacum (2n = 48; SSTT genome) and N. suaveolens (2n = 32). Our objective was to identify a N. tabacum chromosome that carried gene(s) inducing hybrid lethality in this cross. We crossed 10 monosomic lines (2n = 47; Haplo-M to Z, except for Haplo-p and Haplo-V) of N. tabacum to N. suaveolens and produced hybrids by test-tube pollination and ovule culture using synthetic media. We pollinated 178 placentae (13 to 62 per cross), cultured 5215 ovules (136 to 840 per cross), and produced a total of 85 seedlings. Of the 85 seedlings, 81 died before the cotyledonary and leaf-expansion stages of growth, while 4 from a cross to Haplo-Q produced viable hybrids. Three plants grew to maturity and flowered but one plant was infected by fungi and died at the young stage. Chromosome counting, RAPD and LSC analyses revealed that 3 viable plants were true F1 hybrids. These results suggested that the Q chromosome of the S genome harbored a gene or genes that induced non-viable seedlings from a cross between N. tabacum and N. suaveolens.