Fusarium crown and root rot of tomato (Lycopersicon esculentum) caused by Fusarium oxysporum f.sp. radicis-lycopersici (FORL) is a damaging disease of greenhouse crops worldwide. Here, we characterize in vivo, a new strain of FORL (27.2) isolated from wilting, sensitive tomato cultivar Kardelen F-1, widely grown in Turkey. Compared to a reference strain (usaFORL), 27.2 strain was less virulent in terms of disease severity, and plant growth parameters. To determine whether the effects of both strains involve expressions of defence-related genes, we performed reverse transcription-polymerase chain reaction analysis in tomato roots inoculated with strains 27.2/usaFORL. Our results showed that PR1, PR6, and CH9 expressions were upregulated by usaFORL. PR4 expression in plants infected with both strains was downregulated, indicating that systemic acquired resistance (SAR) was not associated with PR4 activation. No transcripts for CH3 were observed after inoculating with both strains, suggesting that CH3 expression may not be involved in defence against FORL. ET-regulated gene (ETR1, ACO1, ACO2, and ACO3) expressions were increased only by usaFORL, indicating their correlation with pathogen virulence, and in turn, reduced ethylene sensitivity and subsequent necrosis. However, both strains downregulated ACO4 transcripts. Finally, both strains induced Pal expression, indicating gene involvement in FORL-induced SAR in tomato seedlings.