Dry fruits of wolfberries (Lycium barbarum L.) are widely used as functional food in Southeast Asia. Recently it is popular in the emerging market such as North America and Europe because of its healthpromoting properties, such as antioxidant, immunomodulatory and neuroprotective. It is a great potential to develop it into a novel functional fresh fruit in the near future. However, basic genetic studies on the nuclear DNA content and karyotype of many Lycium species are not available and might hinder the breeding of novel wolfberry cultivars. The main objective of this study is to estimate the nuclear genome size and karyotype analysis of three Chinese species (L. barbarum, L. chinense and L. ruthenicum) and two species from North America (L. pallidum and L. fremontii). Flow cytometry using woody plant buffer (WPB) indicated that the 2C DNA content of species varied from 4.35 pg (L. barbarum) to 14.94 pg (L. fremontii). The genome size variation of two widely cultivated species in China L. barbarum (4.35 pg/2C) and L. chinense (6.44 pg/2C) suggested different genomic component and structure. The genome size and chromosome counting confirmed that L. fremontii is octaploid (2n = 96). Karyotype analysis indicated that all species shared a highly symmetrical karyotype, suggesting that speciation in Lycium was not accompanied by changes in chromosomal morphology. Our results provided useful information at the genome level of Lycium species.