BACKGROUND: Intestinal microbiota are implicated in risk of necrotizing enterocolitis (NEC) and sepsis, major diseases of preterm infants in neonatal intensive care units (NICUs). Rates of these diseases vary over time and between NICUs, but time and NICU comparisons of the intestinal microbiota of preterm infants are lacking. METHODS: We included 66 singleton infants <29 weeks gestational age with stool samples collected between postnatal days 3 to 21 of life who survived free of NEC and sepsis. Infants were enrolled during 2010 and 2011. Twenty-six infants were enrolled at hospital 1 in Cincinnati, OH, and 40 infants were enrolled at hospital 2 in Birmingham, AL. Samples collected from days 3-9 ("week 1") and days 10-16 ("week 2") were compared between years and hospitals. Microbial succession was compared between hospitals in 28 infants with samples from the first 3 weeks of life. DNA extracted from stool was used to sequence the 16S rRNA gene by Illumina MiSeq using universal primers. Resulting operational taxonomic unit tables were analyzed for differences between years and hospitals using linear discriminant analysis effect size algorithm (LEfSe; significance, p < 0.05). RESULTS: Significant variation was observed in infant microbiota by year and hospital. Among hospital 1 infants, week 1 samples had more phylum Firmicutes (class Bacilli, families Clostridiaceae and Enterococcaceae) in 2010 and more phylum Proteobacteria (family Enterobacteriaceae) in 2011; week 2 samples did not significantly vary over time. However, among hospital 2 infants, the week 1 shift was nearly opposite, with more Proteobacteria (Enterobacteriaceae) in 2010 and more Firmicutes (Bacilli) in 2011; week 2 samples exhibited the same pattern. Regression analysis of clinical covariates found that antibiotic use had an important influence but did not explain these observed shifts in microbiota over time and between hospitals. Microbial succession also differed by hospital, with greater change in microbiota in hospital 1 than hospital 2 infants (p < 0.01, Jaccard distance). CONCLUSION: Colonizing microbiota differ over time and between NICUs in ways that could be relevant to disease. Multi-site, longitudinal studies are needed to reliably define the impact of intestinal microbiota on adverse outcomes of preterm infants.