Citrus is the second most important fruit crop in Colombia after bananas, with production taking place in 26 of the country’s 32 departments. Oranges, sour limes, and mandarins are the most economically important crops in terms of area planted and production. Microsatellites were used to assess the genetic differentiation of oranges, mandarins, tangelos, grapefruits, and acid limes from the Colombian Agricultural Research Corporation AGROSAVIA germplasm bank and collection of micro-grafted plants at Palmira Research Center in Colombia. A total of 121 samples from eight citrus groups were analyzed with 30 fluoromarked simple sequence repeats (SSR) microsatellites. The mean expected heterozygosity and mean observed heterozygosity were 0.58 and 0.57, respectively, and the coefficient of genetic differentiation was 0.558, confirming very high genetic differentiation among the citrus groups evaluated. Microsatellites mCrCIR01B02, AMB5, Ci01C09, mCrCIR08B08, and Ci01C07 were the most informative, presenting a high number of alleles and polymorphic loci percentages of more than 45 %; in addition, they allowed the identification of unique alleles, which can be used to establish the genetic fingerprint of citrus. Genetic differentiation was achieved for seven out of the eight groups evaluated. The SSRs used failed to differentiate the orange groups, possibly due to genetic origin; for this group, other molecular markers are recommended.