Leaky gut, or intestinal barrier immaturity with elevated intestinal permeability, is the proximate cause of susceptibility to necrotizing enterocolitis in preterm neonates. We recently revealed intestinal barrier maturation was associated with exclusive breastfeeding, less antibiotic exposure, most importantly, altered composition of the gut microbiota. However, sequencing short regions of 16S rRNA gene amplicon failed to identify the specific bacterial groups associated with improved or aberrant intestinal permeability. In this study, we performed high-throughput amplicon sequencing of the full length 16S rRNA gene with single-nucleotide resolution for a cohort of 66 preterm neonates born at 24-33 weeks of gestation who had stool collected daily for 21 postnatal days. We assessed their intestinal permeability by measuring urine non-metabolized sugar probes lactulose and rhamnose during the first 7-10 days of life. We observed that intestinal barrier maturation was positively correlated with changes in specific amplicon sequence variants of species of Clostridiales and Bifidobacterium, while leaky gut was associated with specific strains of Escherichia coli. These results are promising in that they support the use of stool microbial biomarkers for the rapid, non-invasive, and cost-effective assessment of intestinal maturation in neonates.