With two sets of wheat near-isolines having different genetic background as test materials, this paper studied the dynamics of their DIMBOA (2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one) contents during Puccinia striiformis f. sp. tritici infection. It was revealed that in un-inoculated plants, no significant difference was observed in DIMBOA contents among various resistant and susceptible inbred lines within the same set. But, at the initial stage of infection, the DIMBOA contents in all resistant isolines displayed a quicker and greater increase, resulting in a significantly higher content of DIMBOA in resistant isolines than in susceptible one throughout the period of disease development. The infection ultimately led to a decline of DIMBOA contents in all infected plants, but the decrement was significantly less in resistant lines than in susceptible one. It could be concluded that DIMBOA was associated with the monogenic resistance to Puccinia striiformis f. sp. tritici in wheat. The study of DIMBOA dynamics in plants suffered from diseases and pests would be useful for understanding the mechanism of resistance, and the screening and breeding of crop varieties with multiple-resistance.