Andean blackberry (Rubus glaucus Benth.) is an emerging fruit crop with significant commercial potential. Despite its growing popularity, basic research about its genetic resources and breeding remains insufficient. The aim of this study was to assess the genetic diversity of Andean blackberry cultivars and related berries species from the main production areas in Ecuador. We analysed a total of 106 samples and performed DNA screening with different molecular markers: random-amplified polymorphic DNAs (RAPDs), inter-simple sequence repeats (ISSRs) and a set of representative samples with amplified fragment length polymorphisms (AFLPs). The tested RAPD primers did not reveal any differentiation among accessions identified as R. glaucus, however one ISSR primer was useful to find polymorphisms allowing the selection of 29 accessions for the analysis with AFLP markers. AFLP-M13 technology was used for screen genetic variations among these accessions and eight wild Rubus accessions. We scored 203 bands using five primer combinations; out of these 152 were informative in R. glaucus. AFLP markers clearly distinguish R. glaucus from the screened wild Rubus species, also an unexpected genetic structure was revealed among R. glaucus cultivars. This genetic differentiation and detection of admixed genotypes suggest a possible introgression of wild Rubus species in R. glaucus. Our findings are relevant for blackberry genetic breeding and use of these genetic resources.