Stripe/yellow rust is caused by a biotrophic fungal pathogen known as Puccinia striiformis f. sp.tritici (Pst). RNA seq technique was used to understand the wheat and stripe rust interaction in three different reaction types. Leave samples of three near-isogenic lines (NILs) of wheat showing Resistant/immune (R), Moderately Resistant (MR), and Susceptible (S) (inoculated with Puccinia striiformis race 574232) responses were collected 48 and 72 h after inoculation (hai). Overall, 8,595 DEGs were upregulated and 8,741 were downregulated with high numbers (4,357) of DEGs identified S-R in comparison at 72hai. Subsequent Gene Ontology (GO) enrichment analysis suggests protein phosphorylation, biological process, and cellular process of internal components of membrane enriched in all three comparisons at both time points. DEGs identified as serine/threonine receptor-like kinases, peroxidases, Mitogen-phosphate Kinases (MAPK), Phenylalanine ammonia-lyase (PAL), and Pathogenesis-related protein (PR) were high in resistant NIL in all comparisons at both time points. The results show that although the responses of the near iso-genic lines are similar, it is the magnitude of the response that plays a role in the differences in their resistance to Puccinia striiformis.