OBJECTIVE: There is a paucity of data characterizing the microbiota of the female upper genital tract, which controversially is described as a sterile site. We examine whether the fallopian tube harbours an endogenous microbial community. DESIGN: This prospective study collected from women undergoing total hysterectomy or salpingectomy-oophorectomy. SETTING: Private hospital gynaecology department. PATIENTS: Fallopian tubes were collected from women diagnosed with benign disease or for prophylaxis. INTERVENTIONS: Samples were interrogated for the presence of microbial DNA using a next generation sequencing technology approach to exploit the V5 to V9 regions of the 16S rRNA gene. MAIN OUTCOME MEASURES: The fallopian tube microbiota was characterized using traditional culture techniques and next generation sequencing. RESULTS: Bacteria were isolated from 50% of cultured samples, and 100% of samples returned positive PCR results. Only 68% of the culture isolates could be confidently identified using automated diagnostic equipment in a clinical microbiology laboratory. Monomicrobial communities were identified only for cultured isolates (50%). Pyrosequencing revealed that all communities were polymicrobial. spp. were not present in all groups, nor were they the most dominant isolates. Distinct differences in the microbial communities were evident for left compared to right fallopian tubes, ampulla versus isthmus, pre- and post- menopausal tissue, and in secretory phase fallopian tubes with and without Mirena intrauterine devices (all < 0.05). CONCLUSION: The female upper genital tract is not sterile. Distinct microbial community profiles in the fallopian tubes of healthy women suggest that this genital tract site supports an endogenous microbiota.