Rapidly proliferating cells require large amounts of folate to support efficient DNA replication, repair and methylation indicating the importance of folate in human metabolism. Milk products are good sources of such vitamins which are produced by probiotics. In order to find suitable strains capable of high folate production, isolation and identification of Lactobacilli in traditional fermented milk from two different provinces located in the west of Iran were carried out. Lactobacillus bacteria were isolated according to the ISO 7889 standard procedure. The isolated bacteria were characterized phenotypically and were screened for their ability to produce folate during fermentation of skim milk. Folate production by the selected strains was between 2.8 to 66.6 mu g/l. Two strains with the highest folate production were then selected. The 16SrRNA genes from these two strains were amplified and sequenced and a phylogenetic tree constructed. The sequencing results in combination with phenotypic and biochemical properties showed that both strains were similar to Lactobacillus crustorum. Therefore, two new strains with an ability of high folate production were isolated and identified. These could be used as probiotics in the dairy industry. Hence, exploiting natural food-grade microorganisms for the production of nutritive dairy products is possible.