Abstract: Tomato yellow leaf curl virus and root-knot nematodes cause diseases in tomato that can lead to heavy production losses. Resistance genes against both pathogens are available and used in breeding. Molecular markers for resistance gene alleles greatly enhance selection of resistant plants in breeding. In order to make marker-assisted selection for the most commonly used resistance genes in tomato breeding more effective, we have developed a multiplex Polymerase Chain Reaction (PCR) assay to simultaneously assess the genotype at four resistance loci. For this purpose, we have selected available markers for the tomato yellow leaf curl disease resistance gene loci Ty-2 and Ty-5 and for the root-knot nematode resistance gene Mi-1 and have developed a new marker for the tomato yellow leaf curl disease resistance gene locus Ty-1/3 to be incorporated in a multiplex PCR assay. The assay correctly predicted the genotypes of tomato breeding lines known to be homo-or heterozygous for the resistance or susceptibility alleles at the Ty-1/3, Ty-2, Ty-5 and Mi-1 gene loci. In wild tomato (Solanum chilense) accessions LA1969 and LA1932, the markers failed to identify alleles conditioning susceptibility at the Ty-2 and Ty-5 loci. Replacement of a single marker of the multiplex assay by another marker was possible without affecting the accurateness of the assay, as long as the size differences of the DNA fragments of the markers were sufficiently large. Combining four markers for resistance alleles commonly used in tomato breeding into a multiplex assay offers a significant cost reduction for marker-assisted breeding in tomato.