Aims: To evaluate the esterase phenotype in Lactococcus lactis strains isolated from traditional Tunisian dairy products. Methods and Results: A collection of 55 L. lactis strains isolated from traditional fermented milk products and three reference strains were identified at species and subspecies level using molecular methods targeted to the 16S rRNA gene and to the histidine operon. The genotypic data obtained allowed the identification of the strains as L. lactis ssp. lactis and L. lactis ssp. cremoris with the prevalence of the ssp. lactis. The phenotypic identification based on arginine hydrolysis, the growth at 40°C and in presence of 4% NaCl showed several discrepancy with the identification data based on genotypic analysis. Additional experiments carried out evaluating the esterase electrophoretic patterns revealed four classes of esterases identified on the basis of their electrophoretic mobility and specific activity on α- and β-naphthyl ester of acetate and propionate. Esterase profiles discriminated the strains in two main groups corresponding to the subspecies cremoris and lactis according to a DNA-based identification. Conclusions: The evaluation of esterase activity represents a novel phenotype for the taxonomic discrimination of the L. lactis ssp. lactis and cremoris. Significance and Impact of the Study: Besides the DNA-based techniques that allow the rapid and accurate species/subspecies identification, the electrophoretic esterase profiles of L. lactis strains represents: (i) a new phenotypic tool to understand the physiology and the ecology of this species; and (ii) a new test for the potential selection of flavour producing strains.