Abstract: Fusarium oxysporum is a main agriculture pathogen which is responsible for wilting in cucumber, muskmelon and watermelon. In order to study the genetic relationship and phylogeny of Fusarium oxysporum, 36 isolates were obtained from Liaoning province. The genetic relationship of Fusarium was determined by using EF-1α and β-tubulin DNA sequencing as well as a universally primed polymerase chain reaction (UP-PCR) analyses. The results show that all 36 isolates were identified as being infected with F. oxysporum by using the specific primers FOF1 and FOR1. Based on the combined sequence and UP-PCR data, the 36 isolates and GenBank isolates were classified into three major groups (Lineage1-3). These groups corresponded with the three clades of F. oxysporum. Inspection of the sequence data revealed Lineage 1 and Lineage 2 had similar sequences whereas Lineage 3 was significantly different from the other two. And this finding matched the results of UP-PCR analysis. However, the GenBank isolates were classified into Lineage 1 and none of the three techniques applied in this study provided host-related resolution for the pathotype isolates or the geographic origin of isolates. This study confirmed that phylogenetic groups have little connection with virulent groups.