Objective Microbial invasion of the amniotic cavity is associated with spontaneous preterm labor and adverse pregnancy outcome, and Mycoplasma hominis often is present. However, the pathogenic process by which M. hominis invades the amniotic cavity and gestational tissues, often resulting in chorioamnionitis and preterm birth, remains unknown. We hypothesized that strains of M. hominis vary genetically with regards to their potential to invade and colonize the amniotic cavity and placenta. Study Design We sequenced the entire genomes of 2 amniotic fluid isolates and a placental isolate of M. hominis from pregnancies that resulted in preterm births and compared them with the previously sequenced genome of the Type strain PG21. We identified genes that were specific to the amniotic fluid/placental isolates. We then determined the microbial burden and the presence of these genes in another set of subjects from whom samples of amniotic fluid had been collected and were positive for M. hominis. Results We identified 2 genes that encode surface-located membrane proteins (Lmp1 and Lmp-like) in the sequenced amniotic fluid/placental isolates that were severely truncated in PG21. We also identified, for the first time, a microbial gene of unknown function that is referred to in this study as gene of interest C that was significantly associated with bacterial burden in amniotic fluid and the risk of preterm delivery in patients with preterm labor. Conclusion A gene in M. hominis was identified that is associated significantly with colonization and/or infection of the upper reproductive tract during pregnancy and with preterm birth.