Ureaplasma spp., commonly identified in the vagina/cervix of pregnant women with spontaneous preterm delivery, are the most frequently detected microorganisms in amniotic fluid. To date, 14 U. spp. serotypes have been characterized; however, modern molecular biology methods can distinguish different U. spp genotypes. Considering these factors, a knowledge gap exists regarding the association between U. spp. genotypes and the risk of the ascension of U. spp. from the cervix to the amniotic cavity. To fill this gap, an expanded multilocus sequence-typing scheme of U. spp. was performed to assess the relationship between cervical and amniotic fluid U. spp. in pregnant women with spontaneous preterm delivery. This study included 109 and 69 pregnant women with spontaneous preterm labor (PTL) and preterm prelabor rupture of membranes (PPROM), respectively. U. spp. DNA in cervical fluid was identified in 49% and 55% of the women with PTL and PPROM, respectively. The concurrent presence of U. spp. DNA in amniotic fluid was observed in 17% and 59% of the pregnant women with PTL and PPROM, respectively. Among pregnant women with PTL and PPROM, 38 expanded sequence types of cervical U. spp. were identified. No associations were observed between specific genotypes, subgroups, or clusters of cervical U. spp. and the presence of amniotic fluid U. spp. in pregnant women with spontaneous preterm delivery.