Objective: To evaluate the stress level of mothers of preterm infants with gestational age ≤34 weeks, hospitalized in neonatal intensive care. Method: A cross-sectional study with 74 mothers of premature infants in neonatal intensive care, who answered the “Parental Stress Scale: Neonatal Intensive Care Unit” instrument, validated in Brazil. Results: The mean stress level was 4.41 (± 0.77) and the general stress level was 4.36 (± 0.76), with a significant difference (p <0.001) between the subscale "Alteration in parental roles” and other subscales, meaning that mothers were in a very stressful situation. Conclusion: All items in the subscale "Alteration in parental roles” of the “Parental Stress Scale: Neonatal Intensive Care Unit” were identified as the main source of stress experienced by mothers. This study suggests carrying out further studies with other methodologies to increase the knowledge of maternal stress in the national context, applying care interventions involving the parents.