Understanding the extent and partitioning of crop genetic diversity is necessary for conserving and utilizing their genetic potentials for breeding. In the present study, fluorescence-labeled amplified fragment length polymorphism markers were used to characterize the genetic diversityand relationships of 79 cultivars and also of 122 F1 hybrids which resulted from six kiwifruit interploid crosses. A high level ofmean genetic diversity was detected (Hj > 0.22) for all cultivars investigated, without significant differences among diploids (2x), tetraploids (4x), and hexaploids (6x). This suggested that no significant genetic erosion occurred in these cultivars, which were directly selected from natural resources or created from crosses. The Unweighted Pair Group Method with Arithmetic Mean analysis of the genetic dissimilarity between cultivars showed three main groups mostly based on their three ploidy levels. Among these, the red-fleshed cultivars which were originally derived from ‘Hongyang’ clustered into one subgroup of group I, suggesting their unique genetic background despite they were marked as different cultivars used in the current kiwifruit industry. By analyzing the genetic variation of hybrids with variable ploidylevels, our genetic analyses further revealed that interploid crosses can increase the genetic diversity of F1 offsprings, especially from the parental combinations of 6x–2x and 6x–4x,in which both parents showed significant differences in morphology and genetic backgrounds. Based on these findings, strategies were proposed for the conservation and utilization of the current kiwifruit genetic resources for future breeding programs.