AbstractChemically induced male sterility (CIMS) is a valuable tool for hybrid breeding in crops. In this context, the acetohydroxyacid synthase (AHAS)‐inhibiting imazapyr has been proposed to induce male sterility in Imisun sunflower (Helianthus annuus L.) when applied during early reproductive development in a two‐fold higher than recommended field dose. This study aimed to perform a comparative transcriptomic analysis to find out the molecular bases of the gametocidal effect of imazapyr on two Imisun genotypes differing in their herbicide resistance level: completely resistant and intermediate resistant. The CIMS of treated plants was confirmed at physiological and anatomical levels. A total of 948 differentially expressed transcript‐derived fragments (TDFs) were identified by complementary DNA amplified fragment‐length polymorphism (cDNA‐AFLP) analysis at the three most informative developmental stages of anther development. Several genes related to xenobiotic metabolism and stress, including cytochrome P450 monooxygenases and ATP‐binding cassette transporters, were identified in the completely resistant genotype. Interestingly, a group of genes, including protein kinases, sulfonylurea receptor 1, aspartic signal peptide‐peptidase, and specific membrane transporters, were recognized from the differential TDFs simultaneously detected in both genotypes. Some of these transcripts were validated by quantitative real‐time polymerase chain reaction (qPCR) in both genotypes. Therefore, these transcripts could be exclusively related to the CIMS in sunflower. Based on the results, we suggest a probable model of action for the gametocidal effect of imazapyr. Our research provides new insights into the molecular mechanisms of male sterility induction in sunflower.