Old yellow enzyme (OYE) is a NAD(P)H flavin oxidoreductase that in Trypanosoma cruzi (TcOYE) catalyzes prostaglandin PGF2alpha synthesis and reduction of some trypanocidal drugs. We performed DNA microarray analysis and it revealed that the levels of transcription of the TcOYE gene were six-fold lower in a T. cruzi population with in vitro-induced resistance to benznidazole (BZ) (17LER) than in the wild-type (17WTS). Further we investigated the TcOYE levels in 15 T. cruzi strains and clones that were either susceptible or naturally resistant to BZ and nifurtimox, or had in vivo-selected resistance to BZ. Northern blot and real-time RT-PCR analyses confirmed our finding that TcOYE transcription levels were lower in 17LER than in 17WTS. In contrast, we detected no differences in TcOYE transcription levels between other T. cruzi samples. All T. cruzi strains contained four copies of TcOYE gene, except 17LER that contained only one. A 42kDa TcOYE protein was detected in all T. cruzi strains tested. The expression of this protein was similar for all samples, with the exception of 17LER for which the protein was nearly seven-fold less expressed. The chromosomal location of the TcOYE gene and the polymorphisms detected in TcOYE nucleotide and amino acid sequences of the T. cruzi strains are associated with the zymodeme but not with drug-resistance phenotype. Our data show that one of the mechanisms conferring in vitro-induced BZ resistance to T. cruzi correlates with deletion of copies of the TcOYE gene. In contrast, the in vivo and natural resistance to BZ are mediated by different mechanisms.