© 2016 Ollberding et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Preterm birth (PTB) is the leading cause of infant morbidity and mortality. Genitourinary infection is implicated in the initiation of spontaneous PTB; however, examination of the urinary microbiota in relation to preterm delivery using next-generation sequencing technologies is lacking. In a case-control study nested within the Conditions Affecting Neurocognitive Development and Learning in Early Childhood (CANDLE) study, we examined associations between the urinary microbiota and PTB. A total of 49 cases (delivery < 37 weeks gestation) and 48 controls (delivery ≥ 37 weeks gestation) balanced on health insurance type were included in the present analysis. Illumina sequencing of the 16S rRNA gene V4 region was performed on urine samples collected during the second trimester. We observed no difference in taxa richness, evenness, or community composition between cases and controls or for gestational age modeled as a continuous variable. Operational taxonomic units (OTUs) classified to Prevotella, Sutterella, L. iners, Blautia, Kocuria, Lachnospiraceae, and S.marcescens were enriched among cases (FDR corrected p≤ 0.05). A urinary microbiota clustering partition dominated by S. marcescens was also associated with PTB (OR = 3.97, 95% CI: 1.19'13.24). These data suggest a limited role for the urinary microbiota in PTB when measured during the second trimester by 16S rRNA gene sequencing. The enrichment among cases in several organisms previously reported to be associated with genitourinary pathology requires confirmation in future studies to rule out the potential for false positive findings.