The fungicide JS399-19 is a novel cyanoacrylate fungicide active against Gibberella zeae, and has been marketed in China for control of fusarium head blight (FHB) on wheat. Forty-three isolates sensitive to fungicide JS399-19 were collected from three commercial wheat fields in China. Forty-five isolates resistant to JS399-19, obtained from five sensitive isolates by selection for resistance to JS399-19, were selected. Three sensitivity levels were identified: sensitive (S), moderately resistant (MR) and highly resistant (HR) to JS399-19, based on a previous study. Eight isolates representing the three sensitivity-level phenotypes were randomly selected for a study on the inheritance of JS399-19 resistance by analysing the sensitivity of hybrid F1 progeny. A nitrate-non-utilizing mutant (nit) was used as a genetic marker to confirm that individual perithecia were the result of outcrossing. Five crosses were assessed: S × S, S × HR, MR × HR, HR × HR and MR × S. In crosses between parents with different sensitivity levels, such as S × HR, MR × HR and MR × S, the progeny fitted a 1:1 segregation ratio of the two parental phenotypes. No segregation was observed in the crosses S × S and HR × HR. It was concluded that the MR and HR phenotypes in G. zeae were conferred by different allelic mutations within the same locus. In these isolates, resistance to JS399-19 was not affected by modifying genes or cytoplasmic components.