Molecular genetic studies on the model plant Arabidopsis thaliana often involve multiple rounds of Agrobacterium-mediated transformation. Such procedures require multiple marker genes that would allow for efficient selection of transgenic plants in each cycle of transformation. Here, we report on a selection marker cassette based on a codon-modified glyphosate N-acetyltransferase (GAT) gene whose expression is driven by a powerful EL2Ω promoter. After introduction of the GAT expression cassette into A. thaliana via Agrobacterium-mediated transformation, glyphosate-resistant primary transformants are efficiently selected by glyphosate, either added to the culture medium or by spraying a glyphosate solution onto seedlings grown in soil. Robust glyphosate-resistant phenotypes are always associated with the presence of the GAT cassette. In addition, RT-PCR analysis of T2 transformants has demonstrated that resistance to glyphosate is associated with higher levels of GAT expression. Resistance conferred by GAT is specific to glyphosate and not to other commonly used selection chemical compounds. These results demonstrate the versatility of the GAT cassette suitable for both large-scale, soil-based selection system of transgenic plants as well as their characterization in vitro.