Objective: To describe the vaginal microbiome of women with premature ovarian insufficiency (POI) receiving systemic hormone therapy (HT). Study design: Descriptive cross-sectional study. Methods: Forty women with POI receiving systemic HT for at least 6 months, were included in the study. Vaginal secretion was collected for DNA extraction followed by Pyrosequencing of the 16S rRNA. The samples were pooled into phylogenetic groups (Ravel – I, II, III, IV, V). Results: Women had mean age of 37.13 (± 7.27) years and POI diagnosis at 27.90 (± 8.68) years, a mean HT duration of 8.20 (± 8.73) years. It was observed that 33.4% of the women presented group I flora, with a predominance of L. crispatus; 9% group II flora, with a predominance of L. gasseri; 33.4% group III flora, with predominance of L. iners; 15.2% group IV flora, with a predominance of anaerobic bacteria; and 9% group V flora, with a predominance of L. jensenii. Conclusion: Women with POI receiving HT presented a vaginal microbiome with a predominance of lactobacilli in the composition of the vaginal flora, specifically L. crispatus and L. iners when evaluated by molecular biology through the pyrosequencing of 16S rRNA.