Yoghurt is an important source of Lactobacilli and other lactic acid bacteria. In Iran, native yoghurts naturally contain some valuable probiotic lactobacilli. The aim of this study was to isolate and identify Lactobacilli strains with high quality probiotic potentials from all kinds of yoghurts made by traditional dairy producers of Isfahan, Iran. Lactobacillus strains were isolated from various traditional yoghurts. Then probiotic properties of the selected lactobacilli were determined. Strong acid and bile salt tolerant strains were considered as high quality probiotics, and identified at the species level by biochemical tests and were further identified according to16s rRNA specific sequences. A total of 82 Lactobacillus strains were isolated. Fourteen strains were graded as high quality probiotic lactobacilli (strong acid and bile tolerants). The majority of high quality probitics were sensitive to the most commonly used antibiotics. The phenotypic characterization of high quality probiotics resulted in identification of different lactobacillus species including 3 Lactobacillus casei, 8 Lactobacillus plantarum, and 3 Lactobacillus pentosus. The results of 16s rRNA gene sequencing assay confirmed the biochemical tests. The results indicated that, the Lactobacilli isolated from native yoghurts of Isfahan have great probiotic potentials, and could be used for production of different probiotic products.