Breeding for tetraploid hybrids has become an optimal strategy in grape breeding. In the present study, we deployed 20 morphological traits and 25 simple sequence repeat (SSR) markers to access the genetic diversity of 278 genotypes. These materials contained four grape varieties (Muscat Hamburg, tetraploid Muscat Hamburg, Kyoho and Hybrid F1 Jumei by crossing of Kyoho and tetraploid Muscat Hamburg) and 274 individuals from Jumei's F2 population. The morphological variation analysis revealed the average Shannon information index and the average Nei's genetic diversity index of 1.14 and 0.62, respectively. The coefficient of variation of 14 traits was higher than 20% and significant correlations were found among some characteristics. Prostrate hairs density of lower side of young leaf was significantly positively correlated with Hairs density on internode of shoot (r=0.203) and Prostrate hairs density of lower side of mature leaf (r=0.128). By principal component analysis (PCA) and cluster analysis, the morphological traits showed eight principal components (PC1-PC8) explained approximately 60.99% of the total variance of the 278 genotypes, and the first three major PCs contributed to approximately 30.08%. The two-dimensional graph of PC1 and PC2 directly reflected the similarity between the phenotype of F2 population and its parents. The cluster analysis of 278 materials based on 20 morphological traits resulted in four main groups. Further genetic variation and diversity evaluation in 278 grapes materials was done using 25 SSR markers. 127 bands totally were generated by 25 SSR markers using PCR amplifcation, including 52 polymorphic fragments. SSR analysis indicated, in the population, the average polymorphism information content (PIC) value was 0.26, the average Nei's genetic diversity index was 0.32, and the average Shannon information index was 0.49. The result showed that these SSR markers were able to distinguish genetic variation among genotypes. The SSR cluster analysis based on UPGMA illustrated that the similarity coeffcient ranged from 0.38 to 1.00 and cluster analysis in 0. 47 similarity coeffcient can divide 278 grapes genotypes accessions into five groups These results based 20 morphological traits and 25 SSR markers would be valuable for genetic diversity analysis and helpful in breeding selection. PCA and clustering analysis revealed that most of tetraploid F2 individuals were similar to Jumei (F1). The remaining individuals showed genetic differentiation with their parents, and they served as good materials for future selection and breeding for new tetraploid grape varieties.