Date palm sap (Phoenix dactylifera L.), called Lagmi, is a sugary substrate that readily ferments through the activity of native microflora that consists mainly of yeasts, lactic acid bacteria and acetic acid bacteria (AAB). The aim of this work was to perform a preliminary step in the isolation and identification of AAB species from different samples of palm sap collected in Southern Tunisia, in order to use them as a starter in vinegar production. AAB were isolated on GEY agar from the fresh palm sap (day (D) 0) and following spontaneous fermentation at room temperature after 3 (D3) and 7 days (D7). A preliminary phenotypic identification of 18 isolates was made by means of Gram, catalase, oxidase and ethanol oxidation to acetic acid, and then further to CO2 and H2O. Subsequently, genotypic identification was conducted by sequencing the gene coding for 16SrRNA and a phylogenetic analysis was obtained based on 16S rRNA sequences. Isolates were identified as Acetobacter tropicalis (11 strains), Acetobacter pasteurianus (3 strains), Acetobacter senegalensis (1 strain), Acetobacter indonesiensis (1 strain), Acetobacter cerevisiaie (1 strain), and Gluconacetobacter liquefacienes (1 strain). The application of selected strains as pure starter cultures for the production of vinegar from palm sap is under investigation in our laboratory. Key words: Date palm sap, Lagmi, acetic acid bacteria (AAB), starter, vinegar.