In this paper, we describe the phenotypic and molecular characteristics of two clinically relevant, vancomycin-resistant (VanB), linezolid-resistant Enterococcus faecium isolates. Pyrosequencing showed the G to T single nucleotide polymorphism at bp 2576 in the genes coding for 23S rRNA and was used to quantify the proportion of G to T mutations among six different 23S rRNA genes in E. faecium as a marker for the molecular level of resistance to linezolid. In both isolates, the G to T mutation was found in two of six alleles, and no further mutations in the genes coding for 23S rRNA were found. The dynamic process of linezolid resistance could be demonstrated by the complete reversion of resistant alleles back to only wild type alleles in consecutive isolates of one isolate. Pyrosequencing being used to detect and quantify resistance to linezolid has been proven as a fast and reliable molecular screening method for monitoring linezolid resistance.