Lactobacilli have been associated with the presence and progression of dental caries. Nevertheless, the relation between certain species or genotypes of Lactobacillus and caries is unclear and there are no data available for the Thai population. This study aimed to examine the distribution of species and genotypes of oral Lactobacillus among children with rather high caries prevalence, and to investigate whether certain species or genotypes were more related to caries activity than others. One hundred and sixty-five children were examined for caries status. Saliva samples were collected and the numbers of lactobacilli were counted. A total of 357 Lactobacillus isolates from 59 children were identified to species level by 16S ribosomal RNA genes polymerase chain reaction (PCR) -restriction fragment length polymorphism and 16S ribosomal RNA gene sequencing. Furthermore, 304 isolates from 56 children were genotyped using arbitrarily primed PCR. Significant correlation was found between levels of lactobacilli and dental caries (P < 0.001). Among the 10 identified species of Lactobacillus, L. salivarius was more prevalent in children with moderate to high caries prevalence compared with children with low caries prevalence, while L. fermentum was the most predominant species in all study groups. Moreover, a genetic heterogeneity of Lactobacillus species was found among the children and those with high caries prevalence tended to be colonized with more than one clonal type. In summary, L. salivarius may be a putative caries pathogen among preschool Thai children.