Grapefruit is the fourth economically most important citrus fruit in the world. In this research Inter- Simple Sequence Repeat (ISSR) markers were used to distinguish twenty-nine grapefruit (Citrus paradisi Macf.), five pummelo (Citrus maxima (Burm.) Merr.) and one Citrus hassaku Hort. Ex Tanaka accessions. Twelve ISSR primers produced a total of 100 fragments and 62 of them were polymorphic. The number of average polymorphic fragments per primer was 5.2. The mean polymorphism information content (PIC) was 0.37. The unweighted pair group method arithmetic average (UPGMA) analysis demonstrated that the accessions had a similarity range from 0.79 to 1.00. The accessions were separated into two main clusters; group A with five pummelos and group B with grapefruits. In the pummelo cluster, all pummelos were distinguished whereas in the grapefruit cluster some accessions were not clearly separated. There was a low level of variation in the grapefruits due to their mutation origin.