Abstract: For researching the genetic diversity and phylogenetic relationships of Phytophthora melonis, infected plants with classical symptoms of cucumber phytophthora blight were collected from 5 different cucumber growing areas in Hunan Province. P. Melonis isolated and purified from diseased cucumbers were preliminarily identified by morphological method of observing the sporangia, then ITS and β-tubulin of 22 strains were amplified using PCR technology and sequenced, these sequenced strains were aligned with related sequences downloaded from Genbank with bioinformatics software. The sequence length of ITS-PCR was about 780 bp, and β-tubulin-PCR was about 800 bp. There was similarity between the sequenced strains and the related sequences downloaded from Genbank. According to the phylogenetic analysis of ITS sequence, the sequences used to construct phylogenetic tree were divided into two large clusters: group I and group II, the sequenced P. melonis was located in group I. Since the sources were different, 22 strains from different areas were gathered in different small branches. According to phylogenetic analysis of β-tubulin sequences, the sequences were divided into four big clusters, 22 P. melonis sequences and downloaded P. melonis sequences were in the group I. The results of classification indicated that the two methods were able to reflect the affinity of different phytophthora, and strains isolated from different areas had genetic diversity, but the same source isolates had close genetic relationship.