Modern plant breeding heavily relies on the use of molecular markers. In recent years, next generation sequencing (NGS) emerged as a powerful technology to discover DNA sequence polymorphisms and generate molecular markers very rapidly and cost effectively, accelerating the plant breeding programmes. A single dominant locus, Frl, in tomato provides resistance to the fungal pathogen Fusarium oxysporum f. sp. radicis-lycopersici (FORL), causative agent of Fusarium crown and root rot. In this study, we describe the generation of molecular markers associated with the Frl locus. An F2 mapping population between an FORL resistant and a susceptible cultivar was generated. NGS technology was then used to sequence the genomes of a susceptible and a resistant parent as well the genomes of bulked resistant and susceptible F2 lines. We zoomed into the Frl locus and mapped the locus to a 900 kb interval on chromosome 9. Polymorphic single-nucleotide polymorphisms (SNPs) within the interval were identified and markers co-segregating with the resistant phenotype were generated. Some of these markers were tested successfully with commercial tomato varieties indicating that they can be used for marker-assisted selection in large-scale breeding programmes.