Fusarium proliferatum known as a pathogen of sheath rot disease. This study aims to determine the genetic diversity by rep-PCR and determine the relationship between the molecular diversity data and the in vitro environmental treatment by using light, pH and temperature and rice variety test. The rep-PCR amplification demonstrated that F. proliferatum was categorized into two groups. Based on the mycelial weight and the growth rate parameters, the 12light/12dark, pH 8, and 25 °C sub-treatments showed the highest outcomes, while in continuous light, pH 6, and 25 °C sub-treatments got the highest spore production. The Inpari 30 rice variety showed the highest disease severity results for F. proliferatum infection. Both the results of the F. proliferatum groups did not affect the fungal growth and fungal infection in several tested rice varieties. These findings suggest that each isolate has a different ability to grow, produce mycelium and spores, and infect a specific rice variety.