Eight Random Amplified Microsatellite markers (RAMs) were used to characterize the genetic diversity found in 14 Prunus materials belonging to the deciduous collection of the Pedagogical and Technological University of Colombia. A total of 121 bands were generated: they range from nine for the GT primer to 26 for the ACA primer, and have molecular weights between 100 and 2050 Kb. At a Nei-Li similarity level of 0.75, four groups were formed, according to the characteristics of the fruit. The number of polymorphic loci varied between 8 (GT and AG) and 21 (ACA); the higher levels of heterozygosity were CA (0.43) and CT (0.41). The average value of heterozigosity for the total population was 0.35, much lower than those found in other Prunus species, but higher than other fruit species where RAM markers were used. Therefore, strategies must be deployed for the collections in order to increase genetic variability, such as the introduction of wild or hybrid materials. The RAM technique proved useful as a method for assessing genetic diversity in species of the Prunus genus.