Fusarium wilt of tomato, caused by Fusarium oxysporum f.sp. lycopersici (Sacc.) is one of the major constraints in tomato production in Rajasthan. The present study was undertaken to investigate the cultural, morphological and molecular variability among ten isolates of Fusarium oxysporum f.sp. lycopersici recovered from various locations of Rajasthan. Among the isolates maximum mycelial growth (90 mm) and maximum sporulation (4.6 x 106/mm2) with whitish regular fluffy growth at middle and light yellow-white pigmentation was recorded in Udaipur isolate (UDPA Fo-1). Whereas, isolate CHTT Fo-7 from Chittorgarh showed minimum mycelial growth (72 mm) and (2.6 x 106 conidia/mm2) sporulation and fluffy growth with deep serrated margin and white colony color with creamy-white pigmentation. The size of macro and micro-conidia measured highest in isolate CHTG Fo-8 with 22.6 (22.1-23.4) × 5.72 (5.41-6.01) µm and 5.89 (5.68-6.10) x 3.04 (2.98-3.1) µm length, respectively. The minimum length and width of macro and micro-conidia was measured in isolate UDPA Fo-1 7.8 (7.2-8.4) x 2.17 (1.95-2.4) µm and 4.05 (3.9-4.2) x 1.10 (1.0-1.21) µm respectively. Molecular characterization of the isolates was performed by using reproducibility of PCR amplification. A total of 10 RAPD primers were used and total 121 reproducible bands were amplified, out of which 94 (77.6%) were polymorphic and 27 (22.3%) were monomorphic. Similarity coefficient among the isolates ranged from 1.8 percent (BNSH Fo-9 and PRTB Fo-3) to 68.9 percent (CHTGFo-8 and JPRDFo-5). Genetic similarity co-efficient was relatively low among all the isolates, which ranged from 0.018 to 0.68. The maximum similarity value (0.689) was obtained between isolates CHTG Fo-8 and JPRD Fo-5.