The present study aimed to investigate the prevalence and quantity of Lactobacillus species in the vaginas of healthy Chinese women. Vaginal samples from 92 fertile and 22 postmenopausal healthy Chinese women were analysed using a 16S rRNA gene clone library and species-specific PCR followed by sequencing and real-time PCR. A total of 13 different Lactobacillus species were detected. Species-specific PCR showed that 3 % of the fertile women were colonized by one species of Lactobacillus and 97 % were colonized by two or more species. Among the postmenopausal women, 91 % were colonized by one species of Lactobacillus and 9 % were colonized by two species. In the fertile women, L. iners (82.61 %), L. crispatus (70.65 %) and L. gasseri (67.39 %) were more prevalent than L. jenseniireuteri and L. delbrueckii were not detected, and the other 10 species were detected in just a few samples. The prevalence of these species according to the clone library differed from the prevalence indicated by the species-specific PCR. According to the semiquantitative analysis, the total Lactobacillus DNA concentrations were higher in fertile women than in postmenopausal women. Sixty-one per cent of the fertile women were predominantly colonized by L. iners, 35 % by L. crispatus, and 2 % by L. gasseri. Associations between pairs of Lactobacillus species in fertile women were significant (P,0.05) between the following pairs: L. iners and L. gasseri, L. iners and L. jensenii, L. iners and L. acidophilus, L. gasseri and L. acidophilus, and L. gasseri and L. jensenii. In conclusion, this study provided detailed information on Lactobacillus species colonizing the vaginas of healthy Chinese fertile and postmenopausal women. The study also showed that the diversity of Lactobacillus species in fertile women was higher than in postmenopausal women. According to our study, different techniques, such as species-specific PCR and comparison against a 16S rRNA gene clone library, resulted in different findings regarding species prevalence. These findings highlight the importance of standardization of techniques used for evaluation of bacterial communities. According to our findings regarding species associations, L. iners and L. gasseri may have influences on colonization and proliferation of other vaginal Lactobacillus species.