Introduction: Critically ill neonates are at high risk of developing renal impairment (RI), including acute kidney injury (AKI), which is associated with short- and long-term pathological outcomes and increased mortality. The incidence of acute kidney injury varied by gestational age (GA) group. Aim: Identify laboratory diagnostic criteria of RI in premature newborns (PNs) with different GA who had severe heterogeneous perinatal pathology. Methods: The study was a prospective cohort of patients admitted electively to the III level Neonatal Intensive Care Unit at the Clinical Maternity Hospital 2 (Chernivtsi, Ukraine) between December 2015 and January 2018. One hundred sixty three PNs were treated at the hospital, from which a total of 68 patients were recruited into the study. Inclusion criteria: the GA is more than 24 weeks and less than 37 weeks; birth body weight is more than 500 g and less than 2500 g; presence of clinical signs of severe perinatal pathology. The severity of the perinatal pathology was classified on the basis of the neonatal Therapeutic Intervention Scoring System. The PNs were divided into three groups according to the GA: Group I consisted of 25 newborns born at the GA of 24-31 weeks, Group II – 25 newborns born at the GA 32-33 weeks, Group III – 18 newborns were born in a GA of 34-36 weeks. The levels of sodium, potassium, calcium and chlorine, alanine aminopeptidase, and aspartate aminopeptidase were measured in serum. The levels of creatinine and urea were measured in serum and urine. The concentration of total protein, albumin, α1-microglobulin, and β2-microglobulin was measured in urine. Findings and results: All PNs had signs of severe perinatal pathology and a neonatal Therapeutic Intervention Scoring System score of 10 or higher during the early neonatal period. There was no statistically significant difference in most serum biochemical parameters between groups of the study, excluding the level of serum chlorine. Our results demonstrated no significant difference in levels of urinary total protein and albumin between groups of the study, but showed a progressive increase the level of urinary creatinine and urea with increasing GA. The maximum level of urinary β2-microglobulin was found in “Late-Preterm” newborns who were born at 34-36 weeks, the minimal level of urinary α1-microglobulin was detected in patients with GA 32-33 weeks. Conclusions: Our results demonstrated that most PNs with severe perinatal pathology have some aspects of impaired tubular and glomerular functions. This is evidenced by changes in urinary new biomarkers. We found direct correlations between GA and serum creatinine (r=0.31, p<0.05), urinary creatinine (r=0.40, p<0.05), urinary α1-microglobulin (r=0.37, p<0.05), and urinary β2-microglobulin (r=0.51, p<0.05). Longer longitudinal cohort studies on PNs are required to determine the prognostic and diagnostic criteria of RI in these patients.