Background: Chagas disease (CD), caused by the parasite Trypanosoma cruzi, is a serious public health concern in Latin America. Nifurtimox and benznidazole (BZ), the only two drugs currently approved for the treatment of CD, have very low efficacies in the chronic phase of the disease and several toxic side effects. Trypanosoma cruzi strains that are naturally resistant to both drugs have been reported. We performed a comparative transcriptomic analysis of wild-type and BZ-resistant T. cruzi populations using high-throughput RNA sequencing to elucidate the metabolic pathways related to clinical drug resistance and identify promising molecular targets for the development of new drugs for treating CD. Methods: All complementary DNA (cDNA) libraries were constructed from the epimastigote forms of each line, sequenced and analysed using the Prinseq and Trimmomatic tools for the quality analysis, STAR as the aligner for mapping the reads against the reference genome (T. cruzi Dm28c-2018), the Bioconductor package EdgeR for statistical analysis of differential expression and the Python-based library GOATools for the functional enrichment analysis. Results: The analytical pipeline with an adjusted P-value of < 0.05 and fold-change > 1.5 identified 1819 transcripts that were differentially expressed (DE) between wild-type and BZ-resistant T. cruzi populations. Of these, 1522 (83.7%) presented functional annotations and 297 (16.2%) were assigned as hypothetical proteins. In total, 1067 transcripts were upregulated and 752 were downregulated in the BZ-resistant T. cruzi population. Functional enrichment analysis of the DE transcripts identified 10 and 111 functional categories enriched for the up- and downregulated transcripts, respectively. Through functional analysis we identified several biological processes potentially associated with the BZ-resistant phenotype: cellular amino acid metabolic processes, translation, proteolysis, protein phosphorylation, RNA modification, DNA repair, generation of precursor metabolites and energy, oxidation-reduction processes, protein folding, purine nucleotide metabolic processes and lipid biosynthetic processes. Conclusions: The transcriptomic profile of T. cruzi revealed a robust set of genes from different metabolic pathways associated with the BZ-resistant phenotype, proving that T. cruzi resistance mechanisms are multifactorial and complex. Biological processes associated with parasite drug resistance include antioxidant defenses and RNA processing. The identified transcripts, such as ascorbate peroxidase (APX) and iron superoxide dismutase (Fe-SOD), provide important information on the resistant phenotype. These DE transcripts can be further evaluated as molecular targets for new drugs against CD.