Non-target-site-resistance (NTSR) to herbicides is a major cause for chemical control failure in a number of weeds. Studies investigating NTSR essentially considered grass weeds. Here, we investigated the transcriptomic bases of NTSR to ALS inhibitors in the global, allergenic weed Ambrosia artemisiifolia. Our objectives were to establish a transcriptome resource for A. artemisiifolia and to use a comparative, quantitative transcriptome sequencing approach (RNA-Seq) to identify candidate NTSR genes in this species. Using the PacBio sequencing technology that generate long sequence reads, a transcriptome resource was obtained from the apical part of one 4-leaf plant, i.e., the developing tissues where ALS inhibitors exert most of their effect. BUSCO analysis of the 51,243 contigs (110.5 Mb) indicated a completeness of 80.2% for the assembly, with a N50=2,427 nucleotides. RNA-Seq was performed on 4 pools of three untreated plants each. Plants in one pool were all resistant (4 pools) or sensitive (4 pools) to imazamox, one major ALS inhibitor used against A. artemisiifolia. Expression levels of all contigs in the transcriptome database were monitored and compared among pools. Sixty-two annotated candidate NTSR contigs were identified on the basis of a significantly different expression in the resistant pools compared to the sensitive pools. They included 5 P450s, 14 glycosyl-transferases, 3 glutathione-S-transferases, 4 ABC transporters and 36 other various secondary metabolism enzymes. Candidate contig expression was then measured individually in 384 plants which sensitivity to imazamox had previously been characterised. This allowed to check candidate contig link with NTSR to imazamox in order to identify NTSR markers. The direct role of the NTSR markers in resistance needs to be assessed by functional validation, and the mutations at the root of the differences in expression observed remain to be identified