Bacitracin is used both in clinical practice and as an animal growth promoter in some countries. Recent reports revealed the dissemination of high-level bacitracin resistance (HLBR) in the genus Enterococcus. The bcrABD operon confers HLBR in an Enterococcus faecalis strain, and the presence of the regulator BcrR was stated as essential for this phenotype. Our objectives were to investigate the spread of the bcrABDR genes in the genus Enterococcus and to evaluate the role of this genotype in the HLBR phenotype. Fifty-eight enterococcal isolates from different environments and species were screened for the presence of the bcrABDR genes, and minimum inhibitory concentrations for bacitracin were determined. Reverse transcriptase polymerase chain reaction (RT-PCR) was used to study the expression of bacitracin resistance genes in strains with HLBR following growth in the presence and absence of the antibiotic. The bcrABDR genotype was found associated with E. faecalis strains from clinical settings bearing a HLBR phenotype. The most disseminated mechanism of bacitracin resistance was the BcrAB ABC transporter, encoded by the bcrAB genes. This transporter, which is constitutively expressed in the absence of bcrR, was sufficient to confer HLBR. Our findings stress the need for further evaluation of bacitracin resistance in this genus.