Fusarium crown and root rot of tomato (Solanum lycopersicum) is the disease caused by the fungal pathogen Fusarium oxysporum f.sp. radicis-lycopersici (FORL). The most effective way to control this disease is to plant resistant varieties. Markers tightly linked to Fusarium crown and root rot could be used in breeding programs to introgress crown rot resistance into new varieties. In this study, we converted the random amplified polymorphic DNA (RAPD) marker UBC#116, linked to the Fusarium crown and root rot resistance gene (Frl), into a co-dominant sequence characterized amplified region (SCAR) marker. A fragment of about 480 bp, linked to the Frl gene, was polymerase chain reaction (PCR) amplified with the use of the UBC#116 primers, cloned and sequenced. A pair of primers were designed and PCR amplification was performed to develop a new SCAR marker for the Frl gene. The new marker was applied for the analysis of 96 tomato genotypes. The RAPD marker UBC#116 was also used and it revealed that the markers were equivalent to each other. However, the development of the new co-dominant SCAR marker has made marker-assisted selection (MAS) more practical, rapid and efficient.