Aim To describe intestinal microbiota in preterm infants admitted to the Neonatal Unit using molecular techniques, and assess the impact of different conditions. Methods/study design A two year period Descriptive study (2011–2012) of gut microbiota in stools from newborns borns under 35 week gestational age (WGA) at birth, admitted in a Neonatal Care Unit. Stool samples were collected: M1 (meconium), M2 (first week), M3 (first month). 5 groups of bacteria were analysed using qPCR technique: Bacteroides, Bifidobacterium, Escherichia coli, Clostridiums and Lactobacillus . Maternal, perinatal and neonate variables were registered. Statistic programs SPSSvs 20. Results In the first month a marked increase (x120) in Bifidobacterium is observed. The increase of Lactobacillus (x2) and E. coli (x7) is lower. Bacteroides and Clostridium remain stable. Regarding WGA in ≤ 30 vs > 30 we observed: - Bifidobacterial colonisation is delayed at birth (2,35 × 10 4 vs 6,02 × 10 4 cfu/g; p = 0,09) and at one week birth age (3,98 × 10 4 vs 1,49 × 10 6 cfu/g; p = 0,007). - Higher numbers of E. coli from the first sample (4,37 × 10 3 vs 9,07 × 10 2 cfu/g; 1,55 × 10 4 vs 2,93 × 10 3 cfu/g; 2,94 × 10 4 vs 9,69 × 10 3 cfu/g), although no significant statistically differences were detected. Conclusions The number of Bifidobacterium and Lactobacillus in faecal samples is higher than the content in Bacteroides , Clostridium and E.coli . The colonisation process of studied bacteria is delayed in prematures born at lower WGA, except for E. coli .