A group of nine strains isolated on Slanetz-Bartley agar and/or Kanamycin esculin azide agar were isolated as presumptive enterococci during a screening of Enterococcus spp. in surface waters. All analysed strains were clearly separated using rep-PCR with the (GTG)5 primer from all enterococci included in the in-house CCM database but they formed a homogeneous cluster matching Lactococcus lactis subsp. lactis representatives. Further identification using extensive biotyping as well as automated ribotyping with EcoRI (RiboPrinter microbial characterization system) confirmed all strains as L. lactis subsp. lactis. Obtained results showed that L. lactis subsp. lactis can be commonly isolated from different surface waters. These strains can be confused with enterococci due to their good growth on enterococcal selective media and their similarity in key phenotypical tests. (GTG)5-PCR revealed as a reliable and fast method for identification of L. lactis subsp. lactis strains while automated ribotyping with EcoRI proved to be a good tool for intraspecies typing purposes. Financial support from project MSM0021622416 is gratefully acknowledged.