In order to enrich the resource of lactic acid bacteria and to systematically clarify the diversity of lactic acid bacteria in traditional Paraguayan fermented dairy products, lactic acid bacterial strains were isolated, purified and identified from traditional Paraguayan fermented milk by combined use of the traditional pure culture method, 16S rRNA gene sequencing and phylogenetic analysis. A total of 79 strains were isolated from eight samples, which were identified to belong to seven species of four genera. Lactococcus lactis was found to be the dominant strain, accounting for 68.35% of the total isolates.