<title>Abstract</title>Hybrid lethality, meaning the death of F₁ hybrid seedlings, has been observed in many plant species, including <italic>Nicotiana</italic>. Previously, we have revealed that hybrids of the selected <italic>Nicotiana occidentalis</italic> accession and <italic>N. tabacum</italic>, an allotetraploid with S and T genomes, exhibited lethality characterized by the fading of shoot color. The lethality was suggested to be controlled by alleles of loci on the S and T genomes derived from <italic>N. sylvestris</italic> and <italic>N. tomentosiformis</italic>, respectively. Here, we extended the analysis of hybrid lethality using other two accessions of <italic>N. occidentalis</italic> identified from the five tested accessions. The two accessions were crossed with <italic>N. tabacum</italic> and its two progenitors, <italic>N. sylvestris</italic> and <italic>N. tomentosiformis</italic>. After crosses with <italic>N. tabacum</italic>, the two <italic>N. occidentalis</italic> accessions yielded inviable hybrid seedlings whose lethality was characterized by the fading of shoot color, but only the T genome of <italic>N. tabacum</italic> was responsible for hybrid lethality. Genetic analysis indicated that first-mentioned <italic>N. occidentalis</italic> accession carries a single gene causing hybrid lethality by allelic interaction with the S genome.