Background: Calibrachoa Cerv. (ex La Llave & Lexarza) is a genus of the Solanaceae family (La Llave and Lexarza, 1825). This genus has a high ornamental and economic value due to its intrinsic variability and multiplicity of flower colours. In Argentina there are eight native species, and one of them is Calibrachoa caesia. The genetic diversity among 35 accessions of C. caesia, from five departments in the province of Misiones, was analyzed using ISSR markers. Results: Thirteen ISSR primers yielded a reproducible banding pattern, with 701 amplified loci and 98% of polymorphism. The ISSR primers 5 CT, 5 CA, 5 GA, 5 GACA, 3 CAC, 3 TG and 3 TC generated 100% polymorphic patterns. The Rp values ranged from 23.20 to 10.29 for 5 GACA and 3 AG primers, respectively, while the average values for MI and PIC were 0.367 and 0.231, respectively. The more informative primers were 5 GACA and 5 GA, and the less informative was 3 AC. Simple matching coefficient of similarity varied from 0.8875 to 0.6659, indicating high levels of genetic similarity among the genotypes studied. The UPGMA cluster analysis indicated three distinct clusters; one comprised genotypes of the five departments, while the second included individuals from Guaraní and Oberá regions and the third cluster included the San Pedro individuals. The overall grouping pattern is in agreement with principal coordinate analysis (PCoA). Conclusions: The Bayesian cluster analysis revealed structuring of the C. caesia population and two clusters were identified, which correspond to UPGMA major clades. The AMOVA test for all populations showed highest genetic variation within populations (90%), meanwhile the Fst coefficient was 0.098, indicating a medium differentiation between populations. These results showed a great intrapopulation genetic diversity but no significant difference was detected among populations. In this work the use of thirteen ISSR markers, allowed the characterization of every individual examined. Therefore, the possibility of using molecular data for varietal identification is a promising tool for application in future breeding programs in the genus Calibrachoa.