The Tsn1 gene confers sensitivity to the necrotrophic effector (NE) ToxA, which is produced by the pathogens that cause tan spot and Stagonospora nodorum blotch on wheat. Although Tsn1 is a susceptibility gene, it contains resistance gene-like features such as protein kinase, nucleotide binding (NB) and leucine-rich repeat (LRR) domains. Previous research indicated that Tsn1 arose in the diploid B-genome progenitor of polyploid wheat. However, nucleotide variation in Tsn1 is nearly non-existent among polyploids. Here, accessions of Aegilops speltoides (SS genome), a close relative of the B-genome progenitor, were studied to further characterize the structure, function, evolution, and diversity of Tsn1. Multiple plants from each of 123 accessions were evaluated for reaction to ToxA and genotyped for presence of Tsn1. A total of 95 accessions were insensitive to ToxA and null for Tsn1, whereas the remaining 28 harbored Tsn1 alleles and were either sensitive or insensitive to ToxA. Comparative sequence analysis of the 4,473 bp coding region from 15 sensitive Ae. speltoides plants revealed numerous single nucleotide polymorphisms (SNPs) compared to the Tsn1 allele in the durum wheat variety Langdon. Among Ae. speltoides accessions, there were approximately the same number of nonsynonymous and synonymous mutations, but none of the nonsynonymous changes occurred within the protein kinase, NB, or LRR domains indicating the importance of these domains for Tsn1 function. The diversity in Ae. speltoides allowed us to gain a better understanding of the evolution of Tsn1, and further studies will enhance our understanding of Tsn1-ToxA interactions.