Contamination of paddy seeds (rice with husk) by Fusarium species can cause spoilage and subsequent production of mycotoxins, especially fumonisins that affect human and animal health. A mycological study was conducted to evaluate the natural occurrence of fumonisin B1 produced by Fusarium proliferatum on paddy grown in different geographic regions of Karnataka (India). A total of 65 isolates of F. proliferatum from paddy samples were analysed by polymerase chain reaction (PCR). One set of primers, Fp3-F and Fp4-R was employed to identify the species F. proliferatum, and another set of primers, FUM1 was employed to determine the fumonisin producing ability of the isolates. All 65 isolates of F. proliferatum scored positive with both set of primers, producing amplified products of the expected sizes. Furthermore, thin layer chromatography (TLC) analysis detected fumonisin B1 (FB1) in all of the PCR positive isolates of F. proliferatum.