Genomic data has created a growing demand for tools and methodologies for studying the genes function, which can be realized through loss of function experiments (gene knockout) or by RNA silencing (knockdown). The develop-ment of binary vectors for Agrobacterium tumefaciens mediated transformation (ATMT) has the advantage of being independent of protoplast formation and can be used directly on a wide variety of fungal species and tissue types. The selection of transformants using bialaphos and carboxin has the advantages of low cost in the transformation and availability of different selectable markers, also allowing the analysis of several genes and combination of study by knockout or knockdown, using selectable markers in the same transformant. Thus, this study aimed to build two binary vectors containing reporter gene and selectable markers that confer resistance to carboxin and bialaphos. The cassettes were constructed using the Gateway system to two fragments. The gene encoding the GFP protein and PtoxA and PtrpCpromoters were cloned into pDONR P1-P5R plasmid. Genes that confer bialaphos and carboxin resistance, bar and cbxr respectively, were cloned into pDONR P5-P2 plasmid. The pPGW plasmid was used as des-tination vector. The gfp gene transcription is controlled by PtoxA promoter and the bar and cbxr genes transcriptions are controlled by PtrpC promoter. These binary vectors were named pGWGFP-BAR and pGWGFP-CBXR. The assembly of cassettes was confi rmed by sequencing, and the validation of vectors is being accomplished through transformation (ATMT) with the plant pathogens Mycosphaerella fi jiensis and Fusarium oxysporum f. sp. cubense
