The genotypic and phenotypic variability of 40 Lactococcus lactis isolates obtained from three cheese-making trials of Batzos cheese made one in each, winter, spring and summer was investigated. RAPD-PCR, plasmid profiling and PFGE were used to study the genetic variability and distinguish closely related isolates. Results showed a high degree of heterogeneity among strains. According to PFGE data, all strains except one were clustered together (at a similarity level of approximately 50%) with the L. lactis subsp. lactis reference strain and eleven groups of isolates consisting of 2-8 strains each were distinguished. Plasmid profiling results revealed that there were eight isolates lacking plasmids and nine having unique plasmids. Twenty-three isolates were allocated into six groups. There was an interesting similarity between the plasmid profiling groups and those formed according to PFGE. Clustering of strains according to RAPD-PCR was in agreement with results obtained by both plasmid profiling and PFGE for the majority of the strains. In addition, results obtained by molecular methods indicate a grouping of most of the strains according to the season of cheese production. All strains inhibited the growth of Escherichia coli O157:H7. Their ability to affect the growth of Yersinia enterocolitica, Staphylococcus aureus, Bacillus cereus, Listeria monocytogenes and Enterococcus faecalis was strain dependent. In 42.5% of the isolates high acidifying ability in milk after 24 h was recorded and these were isolates, mainly, from fresh cheese. The 75% of the isolates from winter cheese exhibited higher Lys- than Leu-aminopeptidase activity while the approximately 67% of the isolates from summer cheese showed higher Leu- than Lys-aminopeptidase activity. Their caseinolytic activity after growth in milk for 24 h was significant with preference for alpha(s)-casein degradation. The majority (90%) of the strains formed methanethiol from methionine and this ability was strain dependent. These results suggest that among the wild lactococcal population from Batzos cheese there are interesting strains appropriate to be used as starters for the dairy industry.