Many cultivars of Litchi chinensis were collected in Maejo University. This study aimed to measure genetic diversity of 22 cultivars collected in the university and one cultivar from orchard in Fang district of Chiang Mai province using 25 primers of ISSR (Inter-Simple Sequence Repeat) marker. The results showed that the total number of bands was 130 (average 5.2 bands/primer) and the total number of polymorphic bands was 112 (average 4.48 bands/primer). The similarity coefficient among cultivars varied from 0.378 (between Haeojeen and Komin) to 1.000 (between Honghuai and Samphaothong, Samphaokaeo and Sareakthong, Kwangjao and O-hia). The constructed dendrogram using longan as out group revealed that the genetics of litchi were divided into 3 groups, rather corresponding to cultivated area. All cultivars of group 1 were cultivated in the central part of Thailand, whereas group 2 consisted of cultivars from the central and the northern part of Thailand and all cultivars of group 3 were cultivated in the northern part of Thailand. Moreover, the bands specific to cultivar were found. The 3rd band of primer 817 present only in Brewster, and the 2nd band of primer 881 was found only in Kimjeng.