Previously, we cloned Fhb1 encoding a Histidine-rich calcium-binding protein (His) and found that Fhb1 results from a rare 752 bp deletion involving the 3’ exon of the His-coding gene. We have shown that Fhb1 is a semi-dominant gene and positively regulate FHB resistance (Li et al., 2019). In this study, we generated mutants of Fhb1 through ethyl methanesulfonate (EMS) treatment of two Fhb1 near isogenic lines (NIL) PHR47 and PHR48. From 2338 and 839 M2 families, 13 and 9 HisR mutants were identified, respectively, using TILLING technology. These mutations are all resulting from single nucleotide changes, including four occurring at the 5’ UTR region and 18 at the coding region that resulted in six synonymous mutations and 12 missense mutations. Phenotypic evaluation of these mutants was conducted at M3 or M4 generations in two field trials. The results showed that 18 mutants did not change at the FHB resistance level compared with the wild type, but four of them each carrying a missense mutation became highly susceptible to FHB. The SNPs in these four susceptible mutants co-segregated with the FHB resistance variations. This study supported that correct HisR protein structure is important for the FHB resistance function of Fhb1.